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Objective To assess the prevalence of mycoplasma genitalium (MG) in patients attending sexually transmitted disease (STD) clinic in Guangzhou, and to provide an epidemiological foundation for clinical treatment and laboratory diagnostics. Methods Utilizing real-time polymerase chain reaction (PCR), we analyzed MG DNA in 2, 749 clinical specimens collected from 2, 722 outpatients in the Dermatology Hospital of Southern Medical University from July 2019 to December 2021. Concurrent testing for MG, Chlamydia trachomatis(CT), and Neisseria gonorrhoeae (NG) was performed on 2, 382 of these specimens. Patient data extracted from medical records were used to investigate the correlation between STD symptoms and MG infection. Results The investigation revealed that the overall prevalence of MG infection was 4.4 % among the sampled patients (120 out of 2,722), with a higher prevalence in males (4.9 %, or 87 out of 1, 790) compared to in females (3.5 %, or 33 out of 932). Notably, the prevalence decreased with increasing age. The highest incidence of MG infection was observed in females aged 18 ~ 25 years (6.4 %, or 18 out of 281), while the lowest was in males aged 46 years and above (1.5%, or 5 out of 342), showing a statistically significant variation across age groups (P < 0.05). Among males with urethritis symptoms, MG positive rate was significantly higher at 7.3 % (42 out of 574). The rate of single MG infection was prominent, accounting for 89.9 % (71 out of 79) in MG-positive male patients and 61.5 % (16 out of 26) in MG-positive female patients. Co-infection rate of MG with CT was 1.2 % in females and 0.3 % in males, indicating a significant difference (P < 0.05). Conclusion The findings suggest a relatively high prevalence of MG infection and co-infection with CT among STD clinic attendees in Guangzhou, particularly in the younger demographic. The study underscores the need for early screening and vigilant surveillance of MG to mitigate its transmission among sexually active populations at high risk. [ABSTRACT FROM AUTHOR]

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Mesenchymal-epithelial transition factor (MET) amplification is an important driver of resistance in epidermal growth factor receptor (EGFR) -mutant non-small cell lung cancer (NSCLC), and the combination of MET proto-oncogene (MET) and EGFR-tyrosine kinase inhibitors (TKIs) has shown promise in overcoming this molecularly defined acquired resistance. Emerging data also demonstrate MET amplification as a resistance driver to TKIs-treated anaplastic lymphoma kinase (ALK)-, RET-, and ROSI-fusion NSCLC. Here, we review the literature on recent research progress of MET amplification as a resistance driver to targeted therapy in oncogene-driven NSCLC and summarize the progress of clinical strategies to overcome the resistance mechanism. [ABSTRACT FROM AUTHOR]

Human gene editing technology is a hot spot and focus in the development of biotechnology, but it has also caused controversies over technical risks, genetic biosecurity, ethical dignity of human society and the legality of application, causing people to worry about the application of this technology. Gene editing for reproductive purposes is generally prohibited internationally, and countries have established legal regulatory systems to regulate the application of gene editing technology according to their own conditions. China shall establish a security risk access system for gene editing technology, ensure national biosecurity, establish and improve the system of ethical norms for scientific research, improve the construction of legislative standardization, and provide legal guarantees for the research and application of gene editing technology.

Objective: To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay., Methods: Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay., Results: The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR., Conclusion: The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

Copyright of Journal of Modern Laboratory Medicine is the property of Journal of Modern Laboratory Medicine Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This study aimed to develop a portable, accurate and easy-to-operate scheme for rapid detection of respiratory virus nucleic acid. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the effect of extraction-free respiratory virus treatment reagent (RTU) on viral nucleic acid treatment and the effect of ultra-fast fluorescence quantitative PCR instrument (FQ-8A) on nucleic acid amplification, respectively. RTU and FQ-8A were combined to develop a rapid detection scheme for respiratory virus nucleic acid, and the positive detection rate was judged by C t value using a fluorescence quantitative PCR instrument, and the accuracy of the scheme in clinical samples detection was investigated. The results showed that RTU had comparable sensitivity to the automatic nucleic acid extraction instrument, its extraction efficiency was comparable to the other 3 extraction methods when extracting samples of different virus types, but the extraction time of RTU was less than 5 min. FQ-8A had good consistency in detection respiratory syncytial virus (RSV) and adenovirus (ADV) compared with the control instrument ABI-7500, with kappa coefficients of 0.938 ( P < 0.001) and 0.887 ( P < 0.001), respectively, but the amplification time was only about 0.5 h. The RTU and FQ-8A combined rapid detection scheme had a highly consistent detection rate with the conventional detection scheme, with a sensitivity of 91.70% and specificity of 100%, and a kappa coefficient was 0.944 ( P < 0.001). In conclusion, by combining RTU with FQ-8A, a rapid respiratory virus nucleic acid detection scheme was developed, the whole process could be completed in 35 min. The scheme is accurate and easy-to-operate, and can provide important support for the rapid diagnosis and treatment of respiratory virus.

Artemisia stolonifera is a relative of A. argyi. The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing. This study aims to establish a method to quickly distinguish between them. At the same time, we examined the reasonability and applicability of the specific polymerase chain reaction(PCR) method. The C/T single nucleotide polymorphism was detected at the position 202 of the sequence, based on which specific primers were designed to identify these two species. The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A. argyi and no band for A. stolonifera, which can be used to detect at least 3% of A. argyi samples mixed in A. stolonifera samples. The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A. stolonifera and no band for A. argyi, which can be used to detect at least 5% of A. stolonifera samples mixed with A. argyi. The limit of detection of the established method was 5 ng DNA. The established PCR method can accurately distinguish between A. stolonifera and A. argyi, which provides an experimental basis for the quality control of A. stolonifera and determines whether the herbs are adulterated.

Objective: To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods., Methods: The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis , and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers' urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit)., Results: An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis , and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively ( t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers' urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01)., Conclusions: A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum .

Objective: To develop a polymerase chain reaction(PCR) method for rapid detection of Listeria monocytogenes in oysters without pre-enrichment., Methods: The combination of β-cyclodextrin and bentonite-coated activated carbon was used to remove PCR inhibitors from oyster samples, and the target gene inlB was used for the PCR subsequently. The specificity, sensitivity, and application of the developed method were verified, and the stability and application of the reagents stored under cryopreservation conditions were evaluated., Results: The specificity of the developed PCR method was 100% for the detection of 130 target bacterial strains and 63 non-target bacterial strains. The method reduced the time required for Listeria monocytogenes detection to 4 h without pre-enrichment, and the detection limit was 10 CFU/25 g. The method was consistent with the conventional culture method on the detection rate and viable bacteria detection rate of Listeria monocytogenes in natural oyster samples(the coincidence rate was 100%). Additionally, the reagents could be used normally after storing at-20 ℃ for at least one year., Conclusion: The PCR method developed in this study has high specificity and sensitivity, and can be used for rapid, accurate detection of Listeria monocytogenes in oysters.

Rapid, sensitive and specific detection tools are critical for the prevention and control of infectious diseases. The in vitro nucleic acid amplification assays, including polymerase chain reaction and isothermal amplification technology, have been widely used for the detection of pathogens. Recently, nucleic acid detection-based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) have been developed, which are rapid, highly sensitive, highly specific, and portable. This review describes the classification and principle of CRISPR/Cas systems and their applications in pathogen detection, and discusses the prospects of CRISPR/Cas systems.

Copyright of Chinese Journal of Lung Cancer is the property of Chinese Journal of Lung Cancer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chinese Journal of Lung Cancer is the property of Chinese Journal of Lung Cancer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The rapid global spread of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has introduced various challenges in global public health systems. The poor applicability and sensitivity of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and antigen-based tests, as well as the persistent emergence of SARS-CoV-2 variants with different mutations hinder satisfactory epidemic prevention and control. Therefore, there is an urgent need for diagnostic technologies capable of distinguishing SARS-CoV-2 variants with high sensitivity and low (or no) equipment dependence. Diagnosis based on clustered regularly interspaced short palindromic repeats (CRISPR) has low equipment requirements and is programmable, sensitive, and easy to use. Various nucleic acid detection tools with great clinical potential have been developed for the diagnosis of infectious diseases. Therefore, this review focuses on the reported state-of-the-art CRISPR diagnostic technologies developed for the detection and differentiation of SARS-CoV-2 variants, summarizes their characteristics and provides an outlook for their development.

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.

The aim of this study was to construct a simple, rapid and ultra-sensitive optical biosensing technique based on rolling circle amplification (RCA), and to apply it to multiple detection of drug-resistant genes of mycobacterium tuberculosis. The common mutation sites of isoniazid, rifampicin and streptomycin resistance genes are katG315 (AGC➝ACC), rpoB531 (CAC➝TAC) and rpsL43 (AAG➝AGG). For these three gene sites, from February 2020 to May 2021, in the Department of Laboratory Medicine of the First Affiliated Hospital of Army Military Medical University, the padlock probe (PLP), primers and capture probes were designed. And a solid-phase RCA constant temperature amplification reaction system based on magnetic beads was constructed and the experimental parameters were optimized. The RCA products were accurately captured by the multicolor fluorescent probes (Cy3/Cy5/ROX), and the single-tube multiple detection of three mutation genes was realized. The sensitivity, specificity and linear range of this method were further verified. The results showed that the response range of katG315 in the same reaction system ranged from 1.0 pmol/L to 0.1 nmol/L. The response range of rpoB531 and rpsL43 ranged from 1.0 pmol/L to 50.0 pmol/L and 1.0 pmol/L to 20.0 pmol/L, and the method had good specificity and sensitivity, and could accurately identify single base mutations in mixed targets, with the minimum detection limit as low as 1.0 pmol/L. The recoveries of simulated serum samples were 95.0%-105.2%. In conclusion, the constant temperature amplification multiple detection method constructed in this study can quickly realize the single-tube multiple detection of three drug resistance mutation sites. This technology is low-cost, simple and rapid, and does not rely on large equipment, providing a new analysis method for pathogen drug resistance gene detection.

Objective: To compare the effectiveness of loop-mediated isothermal amplification (LAMP) assay and microscopic examinations for detection of Schistosoma japonicum infections in Oncomelania hupensis in transmission-interrupted regions, so as to provide insights into the optimization of snail surveillance tools in these regions., Methods: Four hilly schistosomiasis-endemic villages where transmission interruption was achieved were selected in Heqing County of Yunnan Province as the study villages, including Xinzhuang and Gule villages in hilly regions and Lianyi and Yitou villages in dam regions. Snail survey was performed by means of systematic sampling combined with environmental sampling in July 2018. All captured snails were identified for S. japonicum infections using microscopy. In addition, 10 to 20 snails were randomly sampled from each snail habitat following microscopy, numbered according to environments and subjected to LAMP assay. The positive rate of settings with S. japonicum -infected snails was compared among villages., Results: A total of 7 949 living snails were captured from 83 snail habitats in 4 villages, and no S. japonicum infection was detected in snails. There were 226 mixed samples containing 1 786 snails subjected to LAMP assay, and positive LAMP assay was found in 3 mixed samples from 3 snail habitats in 2 dam villages. The positive rates of settings with S. japonicum -infected snails were comparable between Lianyi Village (one setting) and Yitou Village (2 set tings) (5.89% vs. 14.29%, P = 0.344). However, the overall positive rate of settings with S. japonicum -infected snails was significantly higher in dam villages (9.67%, 3/31) than in hilly villages (0) ( P = 0.048)., Conclusions: LAMP assay is more sensitive to detect S. japonicum infections in O. hupensis than conventional microcopy method, which may serve as a supplementary method for detection of S. japonicum infections in O. hupensis in high-risk snail habitats in hilly transmission-interrupted regions.

Digital loop-mediated isothermal amplification (digital LAMP, dLAMP) is a novel nucleic acid amplification technique developed in recent years. It divides the target nucleic acid and LAMP reagent into a large number of independent detection regions, and uses a highly active chain replacement DNA polymerase and four specially designed primers for rapid amplification under isothermal conditions, which provides a good platform for quantitative detection of target nucleic acids. The advantages of high accuracy, high sensitivity, absolute quantification, high tolerance to inhibitors and simple instrumentation make the dLAMP technique very promising in molecular diagnosis, especially in rapid detection of pathogenic microorganisms, it shows a good application prospect in the fields of clinical diagnosis, food safety and environmental monitoring. Certainly, the development of dLAMP still faces some challenges, such as how to avoid non-specific amplification in multiple primer designs, multi-target nucleic acids and simultaneous detection of a large number of samples. With the development of dLAMP technology, this technology will greatly enrich the future development of molecular diagnostics. Applying rapid and effective molecular diagnostic techniques to the diagnosis of pathogenic microorganisms has important social significance for disease prevention and control.

Objective: To analyze the pathogenic bacteria and epidemiological characteristics in children with respiratory tract infection in Tianjin area. Methods: Retrospective case analysis was performed on 2 392 hospitalized children in the wards of respiratory diseases, intensive care unit and special care ward of Tianjin Children's Hospital from June 2018 to May 2019. Thirteen pathogenic bacteria in deep sputum and bronchoalveolar lavage fluid samples were detected by loop-mediated isothermal amplification. The laboratory data and clinical characteristics of the infected children were analyzed, and the comparison between groups was performed by t test or χ 2 test. Results: Among 2 392 cases, 1 407 were males and 985 females. There was no significant difference in the detection rate between males and females (72.5% (1 020/1 407) vs .74.2% (731/985), χ 2 =0.87, P =0.35). A total of 1 751 strains and 12 kinds of positive respiratory pathogens were detected, with a detection rate of 73.2%. Among them, 913 (38.2%) strains were Mycoplasma pneumoniae (MP), 514 (21.5%) were Streptococcus pneumoniae (Sp), 381 (15.9%) were Methicillin-resistant Staphylococcus aureus (MRSA) and 279 (11.7%) were Hemophilus influenzae (Hi). There was significant difference in the detection rate of pathogens among different age groups (χ²=83.67, P< 0.01). The positive rate of alveolar lavage fluid group was higher than that of deep sputum fluid group [81.6% (614/752) vs. 69.3% (1 137/1 640), χ 2 =39.89, P <0.01]. The length of hospital stay of children infected with different pathogens was significantly different (all P <0.01). There was significant difference in duration of fever among children infected with different pathogens (χ²=228.69,103.56, 3.96, 27.38,24.50,41.66, all P <0.05). There were 63 (7.7%) cases of atelectasis, 260 (31.9%) cases of pleurisy and 120 (14.7%) cases of pleural effusion in MP children. Children with Sma were most likely to involve the heart system (2/9), and children with Eco infection had a higher incidence of complications such as those of blood (3/19), urinary (2/19), digestive systems(4/19), systemic inflammatory response syndrome and sepsis (1/19). Conclusions: The main bacterial pathogens of respiratory tract infection in children in Tianjin were MP, Sp, MRSA and Hi. It is suggested that clinicians should not only pay attention to the respiratory symptoms of children, but also pay attention to the complications caused by bacterial pathogen infection, so as to prevent the deterioration of the disease and improve the prognosis.

Objective: To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania ., Methods: Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 ( ITS1 ) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti , Toxoplasma gondii , Plamodium vivax , P. ovale , P. falciparum , P. malariae , L. donovani and L. infantum ., Results: After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum , and no cross-reactions were observed with B. microti , T. gondii , P. vivax , P. ovale , P. falciparum or P. malariae . The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA., Conclusions: A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum , which is effective for field screening of leishmaniasis.

Objective: To establish a nucleic acid assay for detection of Paragonimus skrjabini based on the recombinase-aided isothermal amplification (RAA) technique, and to preliminarily evaluate its detection efficiency., Methods: The metacercariae of P. skrjabini , P. westermani and Euparagonimus cenocopiosus were isolated from crabs, and genomic DNA was extracted for molecular characterization. The cytochrome coxidase 1 ( cox1 ) gene sequence of P. skrjabini was selected as the target gene fragment, and the primers and probes were designed, screened and synthesized for RAA assay. The genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province were used as templates for verification of the fluorescent RAA assay. The fluorescent RAA assay was performed to detect different concentrations of plasmids containing target gene fragment and P. skrjabini metacercariae genomic DNA to determine the sensitivity. Fluorescent RAA assay was performed with recombinant plasmids containing P. skrjabini cox1 gene sequences at different concentrations and P. skrjabini genomic DNA as templates to evaluate its sensitivity, and the genomic DNA of P. westermani , E. cenocopiosus , Clonorchis sinensis and Schistosoma japonicum was detected with fluorescent RAA assay to evaluate its specificity., Results: P. skrjabini , P. westermani and E. cenocopiosus metacercariae were isolated from crabs, respectively. Molecular characterization and phylogenetic analysis confirmed their homology with the genes sequences of standard Paragonimus strains in GenBank. A fluorescent RAA assay was successfully established for nucleic acid detection of P. skrjabini , and the genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province was amplified using the fluorescent RAA assay within 5 min, while the negative control was not amplified. If the recombinant plasmid containing P. skrjabini cox1 gene sequences was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 copies/μL, and positive amplification was observed within 5 min. If genomic DNA was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 pg/μL, and all positive amplifications were found within 5 to 10 min. In addition, the fluorescent RAA assay was tested negative for P. westermani , E. cenocopiosus , C. sinensis and S. japonicum ., Conclusions: A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of P. skrjabini , which has potential values in rapid field detection and species identification in freshwater crabs in areas endemic for P. skrjabini .

Nucleic acid testing is the gold standard for diagnosing HCV infection, and it is of great significance to guide the treatment of HCV patients and change the follow-up strategy. In recent years, its detection technology has become increasingly mature. This article summarizes the necessity and current status of nucleic acid testing for hepatitis C through the analysis of domestic and foreign literature, in order to provide reference for the formulation of relevant policies to eliminate hepatitis C in China.

Objective: To develop a rapid test for detection of Schistosoma japonicum specific gene fragments based on the recombinase-aided isothermal amplification assay (RAA) and nucleic acid dipstick test., Methods: The S. japonicum SjG28 gene fragment was selected as the target gene fragment, and the primers and fluorescent probe were designed and synthesized. Then, a S. japonicum nucleic acid dipstick test was established. The sensitivity of this dipstick test was evaluated by detecting different copies of recombinant plasmids containing the S. japonicum SjG28 gene fragment and different concentrations of genomic DNA from adult worms of S. japonicum , and the specificity of the dipstick test was evaluated by detecting the genomic DNA from Clonorchis sinensis , S. mansoni , Ancylostoma duodenale , S. haematobium , Babesia and Paragonimus westermani ., Results: The S. japonicum nucleic acid dipstick test based on the S. japonicum SjG28 gene fragment showed the minimum detectable limit of 10 copies/μL of the recombinant plasmid containing the S. japonicum SjG28 gene fragment and the minimum detectable limit of 1 pg/μL of S. japonicum genomic DNA, and the dipstick assay tested negative for the genomic DNA from C. sinensis , S. mansoni , A. duodenale , S. haematobium , Babesia and P. westermani ., Conclusions: A rapid, simple, and visualized assay is established for detection of S. japonicum specific gene fragments based on RAA and nucleic acid dipstick test.

The genetically modified (GM) maize 'Shuangkang'12-5 has good insect resistance and herbicide tolerance, which is one of the first series of GM maizes obtained a safety certificate in China, and it has broad application prospect in the future. This study established an on-site rapid detection method for GM 'Shuangkang'12-5 based on recombinase polymerase amplification (RPA) technology, which primes and probe were designed according to the specific flank sequence. Then the best combination of primers and probe was obtained through a screeing process. The amplification results of fluorescence RPA can be directly visualized under blue light. The results showed that the visual detection system of GM 'Shuangkang'12-5 with high specificity, and the detection sensitivity of the method could reached 10 copies. Further research found that the RPA amplification system had a wide range of temperature (34℃-46℃). According to this property, the common self-heating warm pastes on the market were used replace the traditional heating instruments to stimulate the RPA.The results showed that the self-heating warm paste meets the temperature requirement of the RPA system. Finally, we combined the self-heating warm pastes with the RPA visual detection system to conduct on-site detection of GM 'Shuangkang'12-5, and compared the results with the detection results of qPCR. The detection showed that the results of on-site visual detection method established in this study were consistent with the detection results of the qPCR. Moreover, the visual detection method was more shorter in time and the final detection result was clear and easy to distinguish. The rapid on-site visual detection method for GM 'Shuangkang' 12-5 established in this study has high specificity, high sensitivity and convenience. It not only meets the needs of on-site rapid detection of GM 'Shuangkang'12-5, but also provides highlight for the development of other on-site rapid detection methods.

Objective: To analyze the monitoring data of Oncomelania hupensi s in the national schistosomiasis surveillance sites of China from 2015 to 2019, so as to understand the changes of Oncomelania snail status in the schistosomiasis-endemic areas of China and to provide the scientific evidence for Oncomelania snail control., Methods: According to the requirements of National Scheme for Schistosomiasis Surveillance in China ( 2014 Edition ), national schistosomiasis surveillance sites were assigned in all schistosomiasis-endemic counties (cities, districts) and the potential endemic counties (cities, districts) in the Three Gorges Reservoir areas, and Oncomelania snail status was monitored according to different epidemic types. In endemic areas, Oncomelania snail survey was performed by means of systematic sampling and environmental sampling, and the occurrence of frames with Oncomelania snails and the prevalence of Schistosoma japonicum infections in Oncomelania snails were calculated, while in potential endemic areas, the risk of imported Oncomelania snails and Oncomelania snails in floating debris were monitored., Results: Oncomelania snail survey was performed covering an area of 116 834.16 hm 2 in the national schistosomiasis surveillance of China from 2015 to 2019, with 35 007.62 hm 2 Oncomelania snail habitats identified. A total of 6 908 292 frames were surveyed during the 5-year period, and there were 364 555 frames detected with Oncomelania snails, with a 5.28% mean occurrence of frames with Oncomelania snails. Among 997 508 living Oncomelania snails captured, no S. japonicum infections were detected, and loop-mediated isothermal amplification (LAMP) assay detected 18 positive mixed Oncomelania snail samples. During the period from 2015 to 2019, 147.20 hm 2 emerging Oncomelania snail habitats were identified, with an overall tendency towards a rise seen in the proportion of emerging Oncomelania snail habitats in plain regions with waterway networks (0.12% to 92.00%), a tendency towards a rise followed by decline seen in marshland and lake regions (0 to 96.72%), and a large fluctuation in hilly regions (0 to 88.49%). A total of 831.10 hm 2 re-emerging Oncomelania snail habitats were found in the national schistosomiasis surveillance sites of China from 2015 to 2019, with an overall tendency towards a rise seen in the proportion of re-emerging Oncomelania snail habitats in marshland and lake regions (16.05% to 79.66%), an overall tendency towards a decline seen in hilly regions (19.25% to 81.00%), and a minor fluctuation in plain regions with waterway networks (1.10% to 10.14%). During the 5-year period from 2015 to 2019, a total of 48 656 kg floating debris were captured in 4 surveillance sites in the Three Gorges Reservoir areas, and 2 204 snails were found, with no Oncomelania snails identified., Conclusions: The areas of Oncomelania snail habitats tended to be stable in the national schistosomiasis surveillance sites of China during the period from 2015 to 2019, however, there was a gradual rise in the area of Oncomelania snail habitats year by year, and LAMP assay identified positive Oncomelania snail samples, suggesting Oncomelania snail control is far from optimistic in China.

Objective: To investigate the distribution characteristics of Oncomelania hupensis in Guangxi Zhuang Autonomous Region, so as to provide insights into the assessment of the risk of schistosomiasis transmission and the scientific formulation of the schistosomiasis surveillance strategy., Methods: From 2015 to 2019, a total of 19 national schistosomiasis surveillance sites were assigned in Guangxi Zhuang Autonomous Region, including 4 fixed sites and 15 mobile sites. Snail survey was performed by means of systematic sampling in combination with environmental sampling, and the infection of Schistosoma japonicum was detected by the crushing method combined with loop-mediated isothermal amplification (LAMP) assay., Results: From 2015 to 2019, snail habitats were detected at areas of 17 040 to 39 527 m 2 , including 6 214 m 2 emerging snail habitats and 16 563 m 2 re-emerging snail habitats. The overall mean density of living snails was 0.019 2 snails/0.1 m 2 and the occurrence of frames with snails was 1.11% in the national schistosomiasis surveillance sites; however, no S. japonicum infection was identified in snails. The area of snail habitats increased by 121.46% in the national surveillance sites in 2019 as compared to that in 2015; however, 50.34% ( Z = -0.422, P > 0.05) and 42.85% ( χ 2 = 130.41, P < 0.01) reductions were seen in the overall means density of living snails and the occurrence of frames with snails. All snail habitats were distributed in the 4 fixed surveillance sites, and were mainly found in ditches, paddy fields and dry lands, with weeds as the primary vegetation type., Conclusions: There are still risk factors leading to re-emergent transmission of schistosomiasis in Guangxi Zhuang Autonomous Region, such as local snail spread, and the monitoring of schistosomiasis remains to be reinforced to further consolidate the achievements of schistosomiasis elimination in the region.

Objective: To construct of a terahertz metamaterial sensing method for detection of microRNA-21 (miRNA-21) based on duplex-specific nuclease triggered rolling circle amplification. Methods: First, a THz metamaterial sensing method was constructed, and verified by polyacrylamide gel electrophoresis and zeta potential; after optimizing the detection conditions of the sensor, different concentrations of miRNA-21 and other different miRNAs were detected. And this biosensor was compared with other miRNA detection methods; finally, the recovery rate of the biosensor was evaluated. Results: Under the optimal experimental conditions, through the dual signal amplification strategy of duplex-specific nuclease (DSN) cycle recognition and rolling circle amplification (RCA), the THz metamaterial sensor has a response range of 10 fmol/L to 10 nmol/L to the target miRNA-21, with a detection limit of 8.49 fmol/L. And the biosensor has good specificity with the ability to recognize the target miRNA-21 from a variety of microRNAs. And the experiment with recovery rate from 94.33% to 115.33% has been further verified in commercial human serum samples. Conclusion: The terahertz biosensor can achieve highly sensitive and specific detection of the target miRNA-21, which proves the potential for label-free diagnosis and early warning of miRNA-related diseases.

HCV infection is of global concern with the characteristic of insidious onset. Laboratory testing stands indispensably for clinical diagnosis with antibody assay and nucleic acid testing the main methods being adopted. For early detection and diagnosis of HCV infection, it is important to choose the best detection method and diagnostic strategy. This paper reviews the HCV nucleic acid testing methods, as well as performance evaluation of reagents and current strategies.

Human papillomavirus (HPV), is a common spherical DNA virus that can lead to six types of cancers later in life, which has recently garnered human's attention. Microchip capillary electrophoresis (MCE) has provided simple, fast, portable, and sensitive HPV typing assay assisted by a variety of signal amplification technologies. This review presents the latest research progress of MCE in routine HPV typing assays, including both of the MCE techniques and MCE combined with the nucleic acid amplification techniques for HPV assay. The introduction on the former part concerns the MCE system, the MCE chips design and electrophoretic separation methods. The typical MCE system includes high voltage power supply, microfluidic chip of separation, sample injection, electrolyte cell, detection unit and so on. Four different chips are reviewed, containing straight separation channel, T-channel, serpentine channel and dual channel, because these microchips are the most used in the last decade. Furthermore, the high integration and high throughput on a single chip are often integrated the sample preparation unit on a chip. The advantages and disadvantages of different designed microchips are introduced at the same time. The separation methods of chip electrophoresis are briefly introduced. With the development and application of MCE for HPV detection, the separation time is greatly shortened from a few hours to several minutes. The review on the second part gives the comments on various kinds of nucleic acid amplification technologies coupled with MCE for HPV assay. Firstly, the comparative analysis is given on the polymerase chain reaction (PCR) combined with MCE, loop-mediated isothermal amplification (LAMP), PCR combined with restriction fragment length polymorphism (RFLP) for HPV DNA detection, and nucleic acid sequence based amplification (NASBA) for the detection of HPV mRNA, nested PCR and so on. Secondly, the reviews on the other methods beside MCE are also summarized, including the PCR coupled with Fourier transform-infrared spectroscopy (FT-IR spectroscopy), nanotechnology, DNA probes combined with electrochemical methods, reductive Cu(Ⅰ) particles catalyzed Zn-doped MoS 2 quantum dots and T7 exonuclease with electrochemiluminescence, LAMP with CRISPR/Cas12a based lateral. In these non-MCE methods, the electrochemical sensing, e. g., impedimetric detection, pulse voltammetry method and flow biosensor, is an ideal method due to its low background signal and excellent time control ability. Finally, although MCE technologies have been developed and the developed instruments are applied in recent years, there are still some challenges in MCE techniques, methods and applications. The first challenge faced in the application of MCE technique in HPV typing assay is that the MCE device can not be well utilized for the detection of HPV with high resolution and high sensitivity, because MCE can not do signal amplification of HPV nucleic acid. The second challenge is that even though some researchers have successfully integrated PCR and MCE on one chip, the technique still faces difficulty for wide application and there is still no really integrated PCR-MCE chip for HPV detection. The third one is the MCE technique is lack for the manufacture of miniaturized and automatic instrument. At the end of review, the authors' insights are given on the development of automatic, fast, high stable and reliable detection in the HPV typing via the portable MCE device.

Objective: To investigate the performance of ZNA(ZIP Nucleic Acid) probes and its application in the quantitative detection of Chlamydia trachomatis (CT)nucleic acid. Methods: Use CT positive plasmids to compare the PCR amplification curves of ZNA probes coupled with different ZIP numbers. Compare ZNA probes with other three sets of probes [namely, 29mer ordinary Taqman probes (long-DNA probe), 20mer ordinary Taqman probes (short-DNA probe) and MGB probes] for stability in PCR amplification curves and repeated freezing and thawing, and the difference in the detection rate of low-concentration plasmids. Use CT positive clinical samples to compare the difference in amplification curves between ZNA probes, long-DNA probe, short-DNA probe and MGB probes, and the detection rate of low-concentration samples. Results: (1) The Ct value and fluorescence value of the probes coupled with 5ZIP units are both better than those coupled with a smaller number of ZIPs. And the difference is biggest when compared with only coupled with 1 ZIP unit: Ct value increased by 1.34 (sensitivity increased by 2.37 times), and fluorescence value increased by 30%. (2) The amplification efficiency of the ZNA probe coupled with 5 ZIPs is 2.14-2.64 times that of the preferred ordinary Taqman probe and MGB probe, and the fluorescence value is 17%-90% higher. (3) The probe freeze-thaw stability results show that the ZNA probe has the best stability, and the lowest concentration of Ct value has the smallest deviation (CV% = 1.4), which is better than the other three sets of probes (CV%=1.7-3.7). (4) Using 35 CT positive clinical samples to compare the PCR amplification performance, compared with other three sets of probes, the amplification sensitivity of ZNA probes was increased by 1.60, 0.99 and 1.06 times respectively. And the results of the consistency analysis of the Ct value show that compared with short-DNA probe and MGB probes, ZNA probes have better detection performance for clinical samples. (5) Use low concentration plasmid template (200, 100, 50 and 10 copies/mL respectively) to compare the amplification sensitivity of the four sets of probes, the detection rate of ZNA probe is the best. Especially, at the lowest concentration 10 copies/mL, the detection rate of the other sets of probes is only 15%-20%, but the ZNA probe is still 30%. (6) In 20 clinical samples with different low concentrations (200, 150, 100, and 50 copies/mL), the detection rate of ZNA probes was the highest, which were 100%, 95%, 90%, and 70%, respectively. Conclusions: Through testing of the amplification efficiency, fluorescence value, freeze-thaw stability, the amplification performance of clinical samples and the detection sensitivity of low-concentration samples, ZNA probes coupled with 5 ZIPs show better performance than ordinary Taqman probes and MGB probes. As a new probe technology with flexible design and easy synthesis, ZNA probe can further improve detection sensitivity of low concentration samples in the field of gene expression.

With the rapid development of molecular biology, the isothermal amplification technique has been used for the nucleic acid detection of parasites and other pathogens due to its high efficiency and rapid and simple procedures, and has become an important tool to promote the field detection and control of parasitic diseases. Recombinase-aided isothermal amplification assay (RAA), a novel isothermal amplification technique, which is simple and easy to perform, rapid for field detection, no need for high-end equipment, and rapid field detection, may amplify the target gene fragments within 5 to 20 min under an isothermal condition (usually 37 to 42 ℃) and achieve a real-time observation of the amplification results. RAA has been successfully employed for the nucleic acid detection of a wide range of parasites and other pathogens to date, and has shown a high sensitivity and specificity. Notably, such an assay is suitable for the large-scale field detection in non-lab environments, and is therefore considered to have a potential value of application in rapid field detections.

Objective: To establish a recombinase-aided isothermal amplification (RAA) assay for the nucleic acid detection of Angiostrongylus cantonensis ., Methods: The internal transcribed spacer-1 ( ITS1 ) gene sequence of A. cantonensis was used as the detection target sequence, and the specific primers and probes were designed and synthesized, followed by screening of the primers and probes with the highest specificity, to establish the basic and fluorescent RAA assay for nucleic acid detection of A. cantonensis . The sensitivity of the fluorescent RAA assay was evaluated by using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A. cantonensis as the template DNA samples, and the specificity of the fluorescent RAA assay was evaluated by using the genomic DNA from A. cantonensis , Schistosoma mansoni , Ascaris lumbricoides , Clonorchis sinensis , Echinococcus granulosus and Ancylostoma duodenale , as well as Pomacea canaliculata and Biomphalaria straminea snail tissues as the template DNA samples., Results: A fluorescent RAA assay was successfully established for nucleic acid detection of A. cantonensis , which achieved real-time amplification of the specific DNA fragment of A. cantonensis within 20 min at 37 ℃. By using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A. cantonensis as the DNA templates, the lowest detection limits of the fluorescent RAA assay were 10 copies/μL of recombinant plasmids and 100 pg/μL of genomic DNA, respectively. The fluorescent RAA assay was negative for detection of the genomic DNA from A. cantonensis , S. mansoni , A. lumbricoides , C. sinensis , E. granulosus , A. duodenale , and P. canaliculata and B. straminea snail tissues., Conclusions: A simple, rapid fluorescent RAA assay has been successfully established, which has a high sensitivity and specificity for the nucleic acid detection of A. cantonensis .

Objective: To establish a nucleic acid assay for detection of Echinococcus granulosus based on recombinase-aided isothermal amplification (RAA) assay., Methods: The 12S rRNA gene of E. granulosus was selected as the target gene, and the specific primers and fluorescent probes for RAA assay were designed, screened and synthesized to establish a fluorescent RAA assay for detection of E. granulosus . The sensitivity of the fluorescent RAA assay was evaluated using different copy numbers of target gene sequence-contained recombinant plasmids and various concentrations of E. granulosus genomic DNA as templates, and the specificity of the fluorescent RAA assay was evaluated using the genomic DNA from E. granulosus , E. multilocularis , Schistosoma japonicum , S. mansoni , Ancylostoma duodenale , Clonorchis sinensis , Taenia saginata , Spirometra mansoni and Taenia solium as templates., Results: A fluorescent RAA assay was successfully established for detection of E. granulosus , which achieved specific amplification of E. granulosus genomic DNA within 20 min at 39 ℃. The lowest detection limit of the fluorescent RAA assay was 10 copies/μL of recombinant plasmids and 0.1 ng/μL E. granulosus genomic DNA, which exhibited a high sensitivity, and the fluorescent RAA assay was all negative for the genomic DNA from E. multilocularis , S. japonicum , S. mansoni , A. duodenale , C. sinensis , T. saginata , Spirometra mansoni and T. solium , which exhibited a high specificity. In addition, this fluorescent RAA assay successfully detected genomic DNA from E. granulosus cysts., Conclusions: A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of E. granulosus .

Objective: To establish a recombinase-aided isothermal amplification (RAA) assay for nucleic acid detection of Schistosoma mansoni ., Methods: The 121 bp highly-repeated sequence of S. mansoni was selected as the target gene fragment to be detected. The primers and fluorescent probes were designed using the Amplfix software, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of S. mansoni genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of S. japonicum , S. haematobium , Ancylostoma duodenale and Clonorchis sinensis to evaluate the specificity., Results: A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of S. mansoni within 20 min at 39 ℃. The minimum detectable limit of the fluorescent RAA assay was 10 copies/μL using recombinant plasmids as templates and 0.1 fg/μL using S. mansoni genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from S. japonicum , S. haematobium , A. duodenale and C. sinensis ., Conclusions: A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of S. mansoni .

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

Objective: To establish a novel nucleic acid assay for detection of Giardia lamblia based on the recombinase-aided isothermal amplification (RAA) assay, and evaluate its sensitivity and specificity for detection of G. lamblia ., Methods: The specific primer sequences and florescent probes were designed and synthesized based on the G. lamblia β-giardin gene as the target gene, and a fluorescent RAA assay was established. The recombinant plasmids at various copies (containing the β-giardin gene target sequence) and the genomic DNA of G. lamblia at various concentrations were used as templates for the fluorescent RAA assay to assess the sensitivity, and the genomic DNA from G. lamblia , Schistosoma japonicum , Clonorchis sinensis , Cryptosporidium parvum , Ascaris lumbricoides , Salmonella and Shigella was used as templates to assess the specificity of the fluorescent RAA assay., Results: A novel fluorescent RAA assay was successfully established for detection of G. lamblia , which allowed the rapid and specific amplification of the target gene fragments at 39 ℃ within 20 min. The sensitivities of the fluorescent RAA assay were 102 copies/μL and 1 pg/μL for detection of the recombinant plasmid and G. lamblia genomic DNA, respectively, and the fluorescent RAA assay was negative for detection of the genomic DNA from S. japonicum , C. sinensis , C. parvum , A. lumbricoides , Salmonella and Shigella , which showed a high specificity., Conclusions: A fluorescent RAA assay, which is simple, sensitive and specific, is successfully established for nucleic acid detection of G. lamblia .

Objective: To establish a rapid nucleic acid detection technique for identification of Echinococcus multilocularis based on the recombinase aided isothermal amplification assay (RAA) and assess its diagnostic efficiency., Methods: The mitochondrial gene sequence of E. multilocularis (GenBank accession number: AB018440) was used as a target sequence. The primers were designed according to the RAA reaction principle and synthesized, and RAA was performed using the generated primers. E. multilocularis genomic DNA at various concentrations and the pMD19-T (Simple) vector containing various copies of the target gene fragment were amplified using RAA to evaluate its sensitivity for detection of E. multilocularis , and RAA was em- ployed to detect the genomic DNA of E. granulosus G1 genotype, Taenia saginata , T. asiatica , T. multiceps , Dipylidium caninum , Toxocara canis , Trichuris trichiura , Giardia lamblia , Fasciola hepatica , Paragonimus westermani , Fasciola gigantica and Clonorchis sinensis to evaluate its specificity. In addition, the optimized RAA was employed to detect nine tissue specimens of E. granulosus -infected animals, 3 fecal samples from E. granulosus -infected dogs and 2 fecal samples from field infected dogs to examine its reliability and feasibility., Results: The established RAA was able to detect the specific target gene fragment of E. multilocularis within 40 min. The lowest detect limit of RAA was 10 pg if E. multilocularis genomic DNA served as a template. If the re- combinant plasmid was used as a template, the minimally detectable copy number of RAA was 104. In addition, RAA was nega- tive for the genomic DNA of E. granulosus G1 genotype, T. saginata , T. asiatica , T. multiceps , D. caninum , T. canis , T. trichiura , G. lamblia , F. hepatica , P. westermani , F. gigantica and C. sinensis . The established RAA was positive for detection of the tissue specimens of infected animals, and simulated and field dog stool samples., Conclusions: A rapid, sensitive and specific RAA is established, which shows promising values in identification of E. multilocularis and gene diagnosis of alveolar echinococcosis.

Objective: To quantitatively evaluate the diagnostic value of variable-temperature and isothermal nucleic acid amplification techniques in the detection of schistosomiasis japonica using a meta-analysis., Methods: The publications pertaining to the nucleic acid detection of schistosomiasis japonica were searched in electronic databases, including Chinese National Knowledge Infrastructure (CNKI), Wanfang Data, PubMed and ScienceDirect, and the compilations and proceedings of schistosomiasis were manually searched. In addition, the citations of publications associated with the nucleic acid detection of schistosomiasis japonica were traced using a document tracing method. The retrieved literatures were screened according to the inclusion and exclusion criteria, and data were extracted from the included literatures. The quality of the included literatures was assessed using the software RevMan version 5.3, and a meta-analysis was performed using the software MetaDiSc version 1.4., Results: A total of 19 publications covering 24 groups of studies were enrolled, including 5 Chinese publications and 14 English publications. There were 17 groups of studies reporting the comparison between the variable-temperature nucleic acid amplification technique and the golden standard, and 7 groups of studies showing the comparison between the isothermal nucleic acid amplification technique and the golden standard. Assessment of the literature quality indicated a minor overall bias of the included literatures, and the Deek funnel plot showed a possible publication bias in the documents reports variable-temperature nucleic acid amplification techniques. There was a heterogeneity caused by non-threshold effect among the studies associated with the variable-temperature amplification technique, and the random effects model was therefore used to combine the effects. The pooled sensitivity and specificity of the variable-temperature amplification technique were 0.81 (0.79 to 0.83) and 0.73 (0.71 to 0.74) for the diagnosis of schistosomiasis japonica, and area under the SROC curve was 0.944 3. There was no heterogeneity among the studies associated with the isothermal amplification technique, and the fixed effects model was therefore used to combine the effects. The pooled sensitivity and specificity of the isothermal amplification technique were 0.96 (0.94 to 0.98) and 0.95 (0.94 to 0.97) for the diagnosis of schistosomiasis japonica, and area under the SROC curve was 0.989 9., Conclusions: Both variable-temperature and isothermal nucleic acid amplification techniques have a high efficiency for the diagnosis of schistosomiasis japonica, and the isothermal amplification technique shows a relatively higher accuracy than the variable-temperature amplification technique.

Objective: To explore the feasibility of high performance liquid chromatography (DHPLC) combined with multiple ligation-dependent probe amplification (MLPA) for the prenatal diagnosis of spinal muscular atrophy (SMA)., Methods: Three families who had given birth to children with SMA type I were subjected to prenatal diagnosis. Peripheral blood samples were collected from the three couples, and 10 mL amniotic fluid was taken for each fetus through amniocentesis at 16-24 gestational week. Following DNA extraction, maternal contamination was excluded by STR analysis. Copy numbers of the SMN genes were detected by denaturing high performance liquid chromatography (DHPLC). Relative copy number of SMN1, SMN2 and reference genes was detected with a MLPA P021 assay kit., Results: The three couples were all found to harbor heterozygous deletion of exon 7 of the SMN1 gene by DHPLC. MLPA analysis also suggested that the three couples were all carriers of SMA mutations. The fetus of family 1 harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus had SMA. The fetus of family 2 also harbored homozygous deletion of exons 7 and 8 of the SMN1 gene, while the copy number of SMN2 gene was normal, suggesting that the fetus was a SMA patient too. The fetus of family 3 harbored heterozygous deletion of exons 7 and 8 of the SMN1 gene, in addition with heterozygous deletion of exons 7 and 8 of the SMN2 gene, suggesting that the fetus was a carrier., Conclusion: DHPLC can effectively screen carriers of SMA mutations. Combined DHPLC and MLPA can provide accurate diagnosis for fetuses with a high risk for SMA.

Objective: To establish a recombinase aided isothermal amplification (RAA) assay for detection of Clonorchis sinensis ., Methods: The 18S ribosomal RNA ( 18S rRNA ) sequence of C. sinensis was used as the target sequence, and specific primers and probes were designed, synthesized and screened to establish a rapid fluorescent RAA assay for the detection of C. sinensis . Then, the sensitivity of the fluorescent RAA assay was evaluated using the recombinant plasmids containing various copy numbers of DNA fragments and C. sinensis genomic DNA at various concentrations, and the specificity of the fluorescent RAA as say was evaluated using the genomic DNA of Ascaris lumbricoides , Echinococcus granulosus , Schistosoma japonicum , Ancylostoma duodenale and S. mansoni as templates. DNA samples were extracted from the feces containing C. sinensis eggs and freshwater fish containing metacercaria for the fluorescent RAA assay, and the performance for detection of C. sinensis -infected samples was preliminarily assessed in the field., Results: A fluorescent RAA assay for detection of C. sinensis was successfully established, which was feasible for specific amplification of C. sinensis genomic DNA at 39 °C within 20 min. The lowest detection limit was 10 copies/μL if the recombinant plasmid containing various copy numbers of DNA fragments was used as a template, and the lowest detection limit was 3 pg/μL if the C. sinensis genomic DNA at various concentrations served as a template. All detections were negative if the genomic DNA of A. lumbricoides , E. granulosus , S. japonicum , A. duodenale , and S. mansoni was used as templates. In addition, the fluorescent RAA assay showed a high performance for the detection of C. sinensis -infected samples in the field, which successfully detected C. sinensis -infected human and rat fecal samples and Pseudorasbora parva samples., Conclusions: A fluorescent RAA assay is successfully established, which is simple, rapid, sensitivity and specific for detection of C. sinensis .

Objective: To explore the preliminary application effect of real-time fluorescence recombinase polymerase amplification (RPA) in the detection of Candida albicans . Methods: (1) Candida albicans standard strain and negative control bacteria of Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Candida glabrata standard strains of respectively 1 mL were collected and their DNA were extracted by yeast/bacterial genomic kit. The specificity of polymerase chain reaction (PCR), real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. (2) One Candida albicans standard strain and one negative control bacteria of Candida glabrata standard strain were collected, resuscitated, and counted. Candida albicans was diluted 10 times to 1×10(7) to 1×10(1) colony-forming unit (CFU)/mL. The DNA of the two bacteria were extracted as experiment (1). The sensitivity of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. The number of cycles for amplification curve to reach the threshold in real-time fluorescent quantitative PCR, and time of appearance of specific amplification curve in real-time fluorescence RPA were recorded and compared with the results in PCR. The detection limit and rate of the above-mentioned 3 methods in detecting Candida albicans were analyzed, and the correlation between concentration of Candida albicans in real-time fluorescence RPA and detection time was analyzed. (3) One standard strain of Candida albicans was collected, and the DNA was extracted as experiment (1) and detected by PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA. The total detection time of the above-mentioned 3 methods was recorded, respectively. (4) The DNA of 31 clinical samples of suspected Candida albicans infection and 1 clinical sample of Candida albicans collected from cotton swab were extracted, PCR and real-time fluorescence RPA were carried out, and the positive detection rates of the above-mentioned methods were calculated. The DNA of the clinical samples with positive results in both PCR and real-time fluorescence RPA were extracted by yeast/bacterial genomic kit, chelex-100 boiling method, and repeatedly freeze-thawing with liquid nitrogen method, and real-time fluorescence RPA and PCR were carried out. The negative control bacteria was Candida glabrata in real-time fluorescence RPA, while negative control bacteria in PCR were the same as experiment (1). The positive results in PCR and real-time fluorescence RPA were observed and time for amplification curve to reach the fluorescence threshold in real-time fluorescence RPA was recorded, respectively. Data were processed with linear correlation analysis and t test. Results: (1) Three methods showed positive results in detecting standard strain of Candida albicans, and none of the 5 negative control bacteria showed positive results. (2) As the concentration of bacterial solution of Candida albicans decreased, the number of cycles for the amplification curve to reach the threshold increased in real-time fluorescent quantitative PCR, the time for appearance of specific amplification curve prolonged in real-time fluorescence RPA, and brightness of the gel strip weakened in PCR. None of the negative control bacteria in the above-mentioned 3 detection methods showed corresponding positive results. The detection limit of Candida albicans in real-time fluorescence RPA, PCR, and real-time fluorescent quantitative PCR was 1×10(1) CFU/mL. There was a significant negative correlation between the concentration of Candida albicans and the detection time in real-time fluorescence RPA ( r =-0.95, P <0.01). The positive detection rates of PCR and real-time fluorescent quantitative PCR for Candida albicans of 1×10(1) to 1×10(7) CFU/mL were 100%. The positive detection rate of real-time fluorescence RPA for Candida albicans of 1×10(1) CFU/mL was 78%, and the positive detection rate of real-time fluorescence RPA for Candida albicans of 1×10(2) to 1×10(7) CFU/mL was 100%. (3) The total time of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA detection for Candida albicans was 133, 93, and 35 min, respectively. (4) The positive detection rate of real-time fluorescence RPA for 31 clinical samples of suspected Candida albicans infection was 32.26% (10/31), which was slightly lower than 35.48% (11/31) of PCR. Eleven clinical samples showed positive results both in real-time fluorescence RPA and PCR detection. No positive result was observed in the negative control bacteria detected both by real-time fluorescence RPA and PCR. The DNA was extracted by yeast/bacterial genomic extraction kit and chelex-100 boiling method for real-time fluorescence RPA detection. The time for the amplification curve to reach the threshold was (438±13) and (462±12) s, respectively, which was close ( t =1.32, P >0.05). The DNA was extracted by repeatedly freeze-thawing with liquid nitrogen method for real-time fluorescence RPA, and the time for the amplification curve to reach the threshold in real-time fluorescence RPA was (584±15) s, which was significantly longer than that in the other 2 methods ( t =7.55, 6.39, P <0.01). Conclusions: Real-time fluorescence RPA has advantages of rapid detection, simple operation, high sensitivity, and good specificity in detecting Candida albicans, which is worthy of clinical application.

Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.

Rapid detection of pathogenic microorganisms is key to the epidemiologic identification, prevention and control of disease in the field of public health. PCR-based pathogen detection methods have been widely used because they overcome the time-consuming issues that traditional culture-based methods required including the limited window required by immunological detection. However, the requirement on precision temperature-controlled thermal cyclers severely limits their use in resource-limited areas. The detection methods of pathogenic microorganisms based on isothermal amplification of nucleic acids are free of dependence on high-precision temperature control equipment, but requirements for nucleic acids extraction, amplification and detection must be defined. In recent years, a number of alternative methods for pathogenic microorganism detection have been developed by combining microfluidic technology with nucleic acid isothermal amplification technology. By designing the chip structures, optimizing the injection modes, and utilizing multiple detection and quantitative methods, the integration of pathogen nucleic acid extraction, amplification and detection is achieved. The method provides advantages of less instrument dependence, decreased operator requirements, smaller sample size, and higher automation which are suitable for the rapid detection of pathogenic microorganisms in various environments. In this review, we summarize several microfluidic detection methods based on nucleic acid isothermal amplification for pathogens including amplification principles, injection methods and detection methods. These methods provide more capability for the rapid screening of pathogenic microorganisms which enhances the management of infectious diseases in the field of public health.

Objective: To investigate the infection of reproductive Mycoplasma genitalium (MG) detected by real-time fluorescence constant-temperature simultaneous amplification and testing (SAT) of nucleic acid among patients present at the clinics of urology, gynecology or venereal diseases., Methods: We retrospectively analyzed the clinical data on 5 711 patients with suspected genitourinary tract (GUT) infection present at the clinics of urology, gynecology or sexually transmitted diseases in Hangzhou Third People's Hospital, Shaoxing People's Hospital and General Hospital of Eastern Theater Command from January 2018 to December 2018. The patients were aged 16－73 (38.77 ± 11.32) years, 3 425 males and 2 286 females. We collected urine samples from 3 666, GUT secretion samples from 2 095, and both urine and GUT samples from 50 of the patients. Using the SAT technique, we detected the infections of MG, Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (NG) in the patients., Results: Of the 5 711 patients, 294 (5.15%) were found MG-positive, with a significantly higher positive rate in the males than in the females (5.96% ［206/3 425］ vs 3.85% ［88/2 286］, P < 0.05). The laboratory results with the urine and GUT secretion samples from 50 of the cases showed a consistency rate of 100%. Simple MG infection accounted for 52.04% in the 294 MG-positive cases, 63.11% in the 206 MG-positive males, and 26.13% in the 88 females, with a significantly higher positive rate in the males than in the females (P < 0.05). MG combined with UU infection had the highest rate among the mixed infections in both the males and females and in those aged ≤20 years, even higher in the females than in the males (P < 0.05) and in the ≤20-year-old males than in the ≤20-year-old females (8.65% ［9/104］ vs 5.13% ［4/78］, P < 0.05). There were statistically significant differences in the MG-positive rate among different age groups (χ2 = 32.74, P < 0.05)., Conclusions: Among patients with suspected GUT infection, the MG-positive rate is higher in men than in women, with MG + UU-positive as the most common mixed infection, and it decreases with the increase of age. The results of SAT of urine and GUT secretion have a high consistency rate.

Objective To explore the clinical value of one-step visualization loop-mediated isothermal amplification(LAMP)in the detection of Mycoplasma pneumoniae(Mp). Methods One-step visualized LAMP,polymerase chain reaction(PCR),and enzyme-linked immunosorbent assay(ELISA)were used to simultaneously detect 108 clinical Mp specimens in children,which included 73 cases of Mp infection diagnosed by PCR and 35 cases of other chronic/acute respiratory tract infections.On the first day of admission,one-step visualization LAMP,PCR(fluorimetric method),and ELISA were used to test the throat swab and serum sample obtained from the same patient,and the Kappa value was calculated.The consistence between LAMP and PCR and that between LAMP and ELISA were compared.On the fifth day of admission,40 patients were resampled and the findings of these three tests on the first day and on the fifth day were compared. Results One-step visualization LAMP had a sensitivity of 100% and a specificity of 94.3%,whereas ELISA had a sensitivity of 65.8% and a specificity of 82.9%.The ratio of Kappa camparing one-step visualization LAMP and PCR was 0.956 and the ratio of Kappa camparing one-step visualization LAMP and ELISA was 0.38.The number of positive specimens detected by LAMP was higher than that by ELISA on the first day. Conclusions One-step visualization LAMP has excellent sensitivity and specificity in detecting early acute Mp infection.It has high consistency with PCR and can be applied to detect Mp.

Objective: To develop a florescent recombinase-aided amplification (RAA) assay for rapid detection of Schistosoma japonicum -infected Oncomelania snails and explore the optimal method for treatment of snail samples., Methods: Snail samples were divided into 3 groups, and each group consisted of 7 subgroups. There were 50 uninfected snails mixed with 1, 2, 3, 4, 5 and 10 infected snails in the 6 subgroups, respectively, and the remaining subgroup contained 100 uninfected snails mixed with 1 infected snails. DNA was extracted from snails in the three groups using a genomic DNA extraction kit following snail crushing and snail shells removal, crude nucleic acid extraction assay following snail crushing and snail shells removal, and crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, and subjected to florescent RAA and PCR as says. The detection results were compared between the two assays., Results: A florescent RAA assay was developed, which completed the detection of S. japonicum -infected snails at 39 ℃ within 30 min. Following DNA extraction from mass snail samples with a genomic DNA extraction kit following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was one infected snail mixed in 50 uninfected snails. Following DNA extraction using crude nucleic acid extraction method following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was 3 infected snails mixed in 50 uninfected snails. Following DNA extraction with a crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, the lowest detection limit of the florescent RAA assay was 10 infected snails mixed in 50 uninfected snails, while the lowest detection limit of PCR assay was 10 infected snails mixed in 50 uninfected snails., Conclusions: A fluorescent RAA assay that is rapid to detect S. japonicum -infected snails in mass snail samples is successfully developed, which is fast, sensitive and easy to perform. Crude nucleic acid extraction following snail crushing and snail shells removal is the optimal method for the treatment of snail samples.

Objective: To understand the immunological and virological characteristics of HIV-1 infected men who have sex with men (MSM) in the acute phase in Tianjin and evaluate the effects of the fourth generation HIV ELISA and the P24 ELISA for acute HIV-1 infected samples. Methods: From October 2015 to October 2016, MSM were recruited through the community-based organizations in Tianjin. All the participants received rapid HIV test, positive samples were confirmed by Western Blot and negative samples underwent pooled nucleic acid testing. The participants with HIV-1 RNA reactive result underwent testing for viral load and T-cell count after second blood collection. Acute HIV-1 infection was defined as negative rapid HIV test result and the positive results of two HIV RNA tests, then the sensitivity were compared between the fourth generation HIV ELISA and the P24 ELISA to detect the initial HIV-1 RNA positive samples. Results: Among 3 016 MSM screened, 193 were positive in rapid HIV test. Western blot testing indicated that 179 cases were HIV positive, 7 cases were HIV indeterminate and 7 cases were negative. Of 2 823 sero-negative cases, 17 were acute HIV-1 infections. The HIV-1 infection rate was 6.53% (197/3 016) and the acute HIV-1 infection rate was 0.56% (17/3 016), with an average viral load of (5.63±1.50) log(10) copies/ml, an average CD(4) count of (442.82±268.17) cells/μl, an average CD(8) count of (1 069.65±668.22) cells/μl and an average CD(4)/CD(8) ratio of (0.49±0.25). Higher viral load, CD(4) and CD(4)/CD(8) ratio were seen in the acute HIV-1 infection group compared with the chronic HIV-1 infection group ( U =148, P <0.01; U =272, P =0.042 and t =3.147, P =0.005). Demographic characteristics were similar between two groups, except the occupation ( χ (2)=11.016, P =0.026). The sensitivity of P24 ELISA was higher than the fourth generation HIV ELISA in the HIV-1 detection for acute infection (Fisher's exact test, P =0.017). Conclusions: MSM are at risk for acute HIV-1 infection. Screening for acute HIV-1 infection with P24 ELISA would increase the sensitivity of diagnosis and reduce HIV transmission in MSM.

Objective: To investigate the distribution patterns of mosquitoes, midges and related arboviruses in Sichuan province. Methods: Blood-sucking insects were collected from houses and pens, using the ultraviolet lights. Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen. All samples were incubated with BHK-21 and C6/36 cells for virus isolation and then detected for their viral genes. Sequences of the virus were identified and analyzed by molecular biological software, such as BioEdit 7.0.5.3, MEGA 6.0. Results: In total, 17 019 mosquitoes from 3 genera and 4 species and 12 700 midges were collected from the southeast regions of Sichuan province in 2016 and 2017. Among them, 79.4% (13 519/17 019) belonged to Culex tritaeniorhynchus with 11.1% (1 897/17 019) as Armigeres subalbatus , 5.5% (930/17 019) were Anopheles sinensis and 4.0% (673/17 019) were Anopheles sinensis 3 virus strains that isolated from Culex tritaeniorhynchus were identified as typeⅠ Japanese encephalitis virus. Seven pools of mosquitoes isolated from Hejiang county were identified Japanese encephalitis virus gene positive through PCR amplification. With 4 pool midges were detected positive for Akabane virus through PCR gene amplification while midges samples didn't have virus isolates. Conclusions: Culex tritaeniorhynchus appeared the predominant species in the southeast regions of Sichuan. Japanese encephalitis virus transmitted by mosquitoes and Akabane virus by midges were prevalent in southeast Sichuan province.