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One of the main tasks of the NBC Protection Troops is accurate and rapid identification of infectious disease causative agents in case of establishing the fact of biological contamination. Different methods based on the analysis of nucleic acids are most preferred for this purpose. Most of them are based on DNA amplification by polymerase chain reaction (PCR). The result is detected by electrophoretic separation of amplification products, as well as by registration of endpoint fluorescent signal (FLASH modification) or in real time (PCR-RT). Other methods of DNA amplification, such as ligase chain reaction (LCR) and isothermal amplification, are also applicable in practice. The article also describes some identification methods based on nucleic acid sequencing: multilocus sequence typing (MLST) method, sequencing of individual genes and complete genome sequencing. It is concluded that the choice of identification method should be based on the goals and objectives, laboratory facilities, availability of trained personnel and funding levels. Despite the fact that the most informative are methods based on sequencing nucleotide sequences, their implementation in the field is difficult so far due to technological requirements.

G-Quadruplex/thioflavin (G4/THT) has become a very promising label-free fluorescent luminescent element for nucleic acid detection due to its good programmability and compatibility. However, the weak fluorescence efficiency of single-molecule G4/THT limits its potential applications. Here, we developed an entropy-driven catalytic (EDC) G4 (EDC-G4) cycle amplification technology as a universal label-free signal amplification and output system by properly programming classical EDC and G4 backbone sequences, preintegrated ligase chain reaction (LCR) for label-free sensitive detection of single nucleotide polymorphisms (SNPs). First, the positive strand LCR enabled specific transduction and preliminary signal amplification from single-base mutation information to single-strand information. Subsequently, the EDC-G4 cycle amplification reaction was activated, accompanied by the production of a large number of G4/THT luminophores to output fluorescent signals. The EDC-G4 system was proposed to address the weak fluorescence of G4/THT and obtain a label-free fluorescence signal amplification. The dual-signal amplification effect enabled the LCR-EDC-G4 detection system to accurately detect mutant target (MT) at concentrations as low as 22.39 fM and specifically identify 0.01% MT in a mixed detection pool. Moreover, the LCR-EDC-G4 system was further demonstrated for its potential application in real biological samples. Therefore, this study not only contributes ideas for the development of label-free fluorescent biosensing strategies but also provides a high-performance and practical SNP detection tool in parallel.

Aspirin (ASP) is currently the drug of choice for antiplatelet therapy. However, approximately 5%–45 % of patients are resistant to ASP and do not achieve the expected result. At present, a few studies have investigated the correlation between ASP resistance (AR) and single-nucleotide polymorphism (SNP). Traditional detection methods are time-consuming and laborious, affecting the accuracy of personalized medicine. This study aimed to establish a new assay to identify four SNPs associated with AR. A large amount of double-stranded DNA was formed after multiple cycles of specific exponential amplification by ligase chain reaction, the specific melting peak of which was visible in the detection curve, with a detection limit of 10−11mol/L. The specificity experiments of different proportions of wild-type and mutant plasmid standards showed that the novel method could detect up to 1 % allele frequency and the specificity was good. Clinical blood samples of 57 patients were tested in this study. The results were consistent with those of sequencing and more accurate and reliable than those of the high-resolution melting method. The technique used in this study was simple, sensitive and specific compared with the traditional method. Statistical analysis revealed that AR was significantly correlated with the rs12041331 site of the PEAR1 gene and the rs1695 site of the GSTP1 gene, providing an important reference value for the study of AR. [Display omitted] • LCR was successfully constructed to detect four kinds of SNPs related to ASP. • LCR assays are more accurate, simpler, more specific and more practical than HRM. • HRM has higher requirements for the operator than LCR. [ABSTRACT FROM AUTHOR]

G1896A switch is one of the hotspots in subjects affected with hepatitis B. This hotspot mutation is observed in all the different spectrum of hepatitis B, and it has a very dangerous and a long lasting effect. The major purpose of the study was to screen G1986A mutations at a large scale and also to establish ligase chain reaction as a mutation testing tool. Polymerase chain reaction (PCR) and Nucleotide Sequencing was done to identify the G1896A mutation in the precore region of the genome. All the 331 HBV positive patients were screened. Almost 29% (24/82) of the cases remarkably had the presence of G1896A mutation confirmed by LCR and direct sequencing.The precore G1896A mutation is responsible for one third of the patients suffering from precore stop codon mutation. It clearly exhibits that LCR is 100% in sync with direct sequencing and is extremely reliable and the results are highly reproducible.

Highly sensitive detection of low-frequency EGFR-L858R mutation is particularly important in guiding targeted therapy of nonsmall-cell lung carcinoma (NSCLC). To this end, a ligase chain reaction (LCR)-based electrochemical biosensor (e-LCR) with an inverted sandwich-type architecture was provided by combining a cooperation of lambda exonuclease-RecJf exonuclease (λ-RecJf exo). In this work, by designing a knife-like DNA substrate (an overhang ssDNA part referred to the "knife arm") and introducing the λ-RecJf exo, the unreacted DNA probes in the LCR were specially degraded while only the ligated products were preserved, after which the ligated knife-like DNA products were hybridized with capture probes on the gold electrode surface through the "knife arms", forming the inverted sandwich-type DNA structure and bringing the methylene blue-label close to the electrode surface to engender the electrical signal. Finally, the sensitivity of the e-LCR could be improved by 3 orders of magnitude with the help of the λ-RecJf exo, and due to the mutation recognizing in the ligation site of the employed ligase, this method could detect EGFR-L858R mutation down to 0.01%, along with a linear range of 1 fM-10 pM and a limit detection of 0.8 fM. Further, the developed method could distinguish between L858R positive and negative mutations in cultured cell samples, tumor tissue samples, and plasma samples, whose accuracy was verified by the droplet digital PCR, holding a huge potential in liquid biopsy for precisely guiding individualized-treatment of NSCLC patients with advantages of high sensitivity, low cost, and adaptability to point-of-care testing.

G1896A switch is one of the hotspots in subjects affected with hepatitis B. This hotspot mutation is observed in all the different spectrum of hepatitis B, and it has a very dangerous and a long lasting effect. The major purpose of the study was to screen G1986A mutations at a large scale and also to establish ligase chain reaction as a mutation testing tool. Polymerase chain reaction (PCR) and Nucleotide Sequencing was done to identify the G1896A mutation in the precore region of the genome. All the 331 HBV positive patients were screened. Almost 29% (24/82) of the cases remarkably had the presence of G1896A mutation confirmed by LCR and direct sequencing. The precore G1896A mutation is responsible for one third of the patients suffering from precore stop codon mutation. It clearly exhibits that LCR is 100% in sync with direct sequencing and is extremely reliable and the results are highly reproducible. [ABSTRACT FROM AUTHOR]

[Display omitted] • We demonstrate simultaneous single-molecule detection of FTO and ALKBH3. • Only a single DNA ligase can achieve multi-cyclic cascade amplification. • This method exhibits high sensitivity and good specificity. • This method can screen inhibitors and detect cellular RNA demethylases. • This method can discriminate breast cancer patients from healthy persons. RNA demethylases play a crucial role in gene expression regulation by controlling RNA methylation levels, and their abnormal expressions are associated with many human diseases. Herein, we develop a single-molecule detection method for simultaneous quantification of N6-methyladenosine (m6A) demethylase FTO and N1-methyladenosine (m1A) demethylase ALKBH3 based on RNA demethylation-directed multi-cyclic ligase chain reaction (LCR) cascades. We design a ssDNA containing m6A modification as the substrate of FTO and a ssDNA containing m1A modification as the substrate of ALKBH3. FTO and ALKBH3 catalyze the demethylation of m6A and m1A to generate normal adenine (A), respectively. Afterwards, Taq ligase initiates the LCR reaction with the demethylated ssDNAs as the templates to produce abundant biotinylated Cy3/Cy5-labeled signal probes. Subsequently, the resultant biotinylated probes are captured by streptavidin-coated magnetic beads (MBs) to form the MB-ssDNA-Cy3/Cy5 complexes. After magnetic separation, the ssDNAs in the MB-ssDNA-Cy3/Cy5 complexes are digested by DNase I, liberating abundant Cy3 and Cy5 fluorophores that can be counted by single-molecule detection. Remarkably, only a single DNA ligase is employed to achieve target signal conversion and multi-cyclic cascade amplification. This method displays extremely high sensitivity with a limit of detection of 6.33 × 10−18 mol/L for FTO and 1.44 × 10−17 mol/L for ALKBH3. Moreover, it can measure the enzyme kinetic parameters, screen inhibitors, and quantify cellular FTO and ALKBH3 activities with single-cell sensitivity. Furthermore, it can differentiate the FTO and ALKBH3 levels in the tissues of healthy human and breast cancer patients, offering a robust platform for biomedical research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized "negative" signal of wobble-base pair-mediated amplification was fully utilized as a "positive" one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue ( I MB ) and current of ferrocene ( I Fc ) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: I MB / I Fc > 2; GG homozygote: I MB / I Fc < 1; GA heterozygote: 1 < I MB / I Fc < 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.

Circulating tumor DNA (ctDNA) in blood carries genetic variations associated with tumors. There is evidence indicating that the abundance of single nucleotide variant (SNV) in ctDNA is correlated well with cancer progression and metastasis. Thus, accurate and quantitative detection of SNVs in ctDNA may benefit clinical practice. However, most current methods are unsuitable for the quantification of SNV in ctDNA that usually differentiates from wild-type DNA (wtDNA) only by a single base. In this setting, ligase chain reaction (LCR) coupled with mass spectrometry (MS) was developed to simultaneously quantify multiple SNVs using PIK3CA ctDNA as a model. Mass-tagged LCR probe set for each SNV including mass-tagged probe and three DNA probes was firstly designed and prepared. Then, LCR was initiated to discriminate SNVs specifically and amplify the signal of SNVs in ctDNA selectively. Afterward, a biotin-streptavidin reaction system was used to separate the amplified products, and photolysis was initiated to release mass tags. Finally, mass tags were monitored and quantified by MS. After optimizing conditions and verifying performance, this quantitative system was applied for blood samples from breast cancer patients, and risk stratification for breast cancer metastasis was also performed. This study is among the first to quantify multiple SNVs in ctDNA in a signal amplification and conversion manner, and also highlights the potential of SNV in ctDNA as a liquid biopsy marker to monitor cancer progression and metastasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

Most of practical methods for detection of single nucleotide polymorphism (SNP) need at least two steps: amplification (usually by PCR) and detection of SNP by using the amplification products. Ligase chain reaction (LCR) can integrate the amplification and allele discrimination in one step. However, the detection of LCR products still remains a great challenge for highly sensitive and quantitative SNP detection. Herein, a simple but robust strategy for real-time fluorescence LCR has been developed for highly sensitive and quantitative SNP detection. A pair of LCR probes are firstly labeled with a fluorophore and a quencher, respectively. When the pair of LCR probes are ligated in LCR, the fluorophore will be brought close to the quencher, and thus, the fluorescence will be specifically quenched by fluorescence resonance energy transfer (FRET). The decrease of fluorescence intensity resulted from FRET can be real-time monitored in the LCR process. With the proposed real-time fluorescence LCR assay, 10 aM DNA targets or 100 pg genomic DNA can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LCR and gives a wide dynamic range for detection of DNA targets (from 10 aM to 1 pM). As LCR has been widely used for detection of SNP, DNA methylation, mRNA and microRNA, the real-time fluorescence LCR assay shows great potential for various genetic analysis. [ABSTRACT FROM AUTHOR]

The global pandemic of coronavirus disease 2019 (COVID-19) has significant impact on the entire human society. However, in the face of continually emerging more contagious SARS-CoV-2 variant, the risk to bog down into more severe crisis is around us anytime. Here, we introduce an isothermal, ultrasensitive method for identifying important SNV mutations of SARS-CoV-2. It is based on combined specificity of t oehold-assisted linear probe l igation and in vitro t ranscription signal enlargement, TLT. A ready-to-use panel of TLT assay is developed including detection of 80 crucial SARS-CoV-2 SNVs, by which people could response to the next coming contagious virus variant more rapidly. These advanced point-of-care features make TLT one good approach for large scale population testing of special SARS-CoV-2 variants of interesting. An isothermal, ultrasensitive method for identifying SNV mutations of SARS-CoV-2, which based on combined specificity of toehold-assisted linear probe ligation and in vitro transcription signal enlargement, TLT. A ready-to-use panel of TLT assay is developed including detection of 80 crucial SARS-CoV-2 SNVs. [Display omitted] • An isothermal, ultrasensitive method for identifying SNV mutations of SARS-CoV-2. • Toehold-assisted linear probe ligation and in vitro transcription signal enlargement, TLT. • Detection of 80 crucial SARS-CoV-2 SNVs. [ABSTRACT FROM AUTHOR]

A gapped ligase chain reaction (gLCR)-based technique was developed and tested on clinical formalin-fixed, paraffin-embedded (FFPE) tissues from colorectal cancer patients. The technique was designed to detect low-level KRAS codon 12 or 13 mutations or confirming doubtful results gained by less sensitive KRAS screening techniques. The gLCR approach was compared with mutation screening techniques commonly used in routine diagnostics regarding sensitivity and specificity. The herein described monoplex gLCR technique is a useful and powerful tool for detecting low-level KRAS codon 12 and 13 mutations in a vast majority of wild type DNA. The gLCR has the capacity to detect one mutated allele in an excess of at least one million wild type alleles (0.0001 %) and is therefore an ideal technique for confirming doubtful KRAS mutation screening results obtained by other techniques. The variance of the gLCRs signal amplitude was very low and is showing a high reproducibility with constant sensitivity even at higher dilutions. The financial effort and the handling time for this technique are low and comparable to a standard cycle sequencing reaction. Additionally, the gLCR technique is easy extendable for the detection of many other clinical relevant mutation hotspots. [ABSTRACT FROM AUTHOR]