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This study intends to describe for the first time a cohort of Mexican patients with Costello syndrome. The five exons of the HRAS gene were amplified in DNA samples from 13 patients with a clinical suspicion of Costello syndrome. PCR products were sequenced using the Ready Reaction Big Dye Terminator v.3.0 Kit and an ABI PRISM 310 sequencer. Only five patients (38%) showed causal variant in codon 12 of the HRAS gene (four with the p.Gly12Ser and one with the p.Gly12Ala variant). Three patients showed silent polymorphic variants (p.His27His and p.Leu159Leu). Clinical features in patients carrying the causal variant were variable. The alternative diagnosis of cardio-facio-cutaneous syndrome was considered in patients who did not have a causative variant in HRAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder caused by FMR1 premutation expansion of CGG repeats. FXTAS can be misdiagnosed with many neurodegenerative disorders manifesting with cerebellar ataxias owing to their overlapping clinical and radiological features. The frequency of the FMR1 premutation allele in Japan has not been fully determined. Herein, we aimed to determine the frequency of FMR1 premutation alleles in Japanese patients with undiagnosed cerebellar ataxia and multiple system atrophy, using repeat-primed PCR in 186 patients with adult onset of undiagnosed cerebellar ataxia and 668 patients with multiple system atrophy, to identify expanded CGG repeats as well as to detect AGG interruptions within the expanded alleles. The size of expansions was estimated using fragment length analysis of PCR products obtained by conventional PCR employing a pair of unique primers flanking the repeat sequence. We identified FMR1 premutation alleles in three male patients. One patient revealed 84 repeat units with one AGG interruption and another patient showed 103 repeat units. Both had presented with sporadic cerebellar ataxia, giving an estimated frequency of 3.7% among Japanese male patients with sporadic cerebellar ataxia with age at onset above 50 years. One patient with the clinical diagnosis of multiple system atrophy harbored 60 repeat units with four AGG interruptions. FMR1 intermediate alleles were observed in two males and one female among the multiple system atrophy patients. We found that genetic tests for FMR1 premutation should be considered in Japanese male patients with cerebellar ataxia with the age at onset above 50 years.

This study was conducted to investigate Babesia parasites infecting sheep in eight districts of Sulaimani governorate/north Iraq from April to October 2017. Forty flocks of small ruminants were selected to collect blood samples randomly from 450 sheep. The samples were examined for babesiosis by microscopic examination and PCR. Primers based on the 18S rRNA were used for Babesia diagnosis, followed by sequencing of the amplicons for confirmation of the PCR product identities. Seventy-four samples (16.44%) showed the presence of Babesia piroplasms microscopically, while 116 (25.78%) samples were positive using PCR. Results showed that B. ovis was reported in 15.78% (n = 71), and B. motasi in 10.0% (n = 45) of the samples. Also, BLAST analysis of the obtained partial sequences of the 18S rRNA gene from current study isolates revealed the existence of both B. ovis and B. motasi, with a high homology degree of nucleotide identity with other nucleotide sequences of Babesia spp. in GenBank database. Distribution of babesiosis, according to the sampling time, revealed that high-frequency rates occur during July and August. Based on the result data, babesiosis was mainly caused by B. ovis and B. motasi.

Background Domestic rabbits (Oryctolagus cuniculi) can develop a variety of cutaneous neoplasms, including squamous cell carcinoma (SCC). A detailed review of gross and microscopic pathology and response to treatment of spontaneously arising SCCs in domestic rabbits has not been published. Methods A retrospective survey study of the clinical characteristics and response to treatment in 39 cases of spontaneous SCC in pet rabbits was performed in an attempt to better characterize the typical presentation, prognosis, and therapeutic response of SCCs in domestic rabbits. Sixteen of these cases were also selected for papillomavirus testing using a generic polymerase chain reaction. Results SCC was identified in rabbits between 2 and 10 years of age, with a median age of 7 years. The neoplasm has a predilection for ears and feet and the conventional subtype is most commonly diagnosed microscopically. Lighter colored rabbits may be predisposed to developing SCC. The majority of cases examined were found in rabbits housed primarily indoors. Only one SCC tested positive for papillomavirus and was located in the oral cavity. Sequencing of the detected PCR product detected 98.75% similarity to human papillomavirus type 120. The significance of this virus for tumorigenesis is unknown. Conclusions and clinical relevance Aggressive surgical resection provided the most successful therapeutic option and proved curative in 12 of 23 rabbits. Papillomavirus likely does not play a major role in the development of spontaneous SCCs in pet rabbits. More research is needed to investigate the use of adjunctive therapies in treatment of this neoplasm in pet rabbits.

Despite scientific progress, the gene sequences for many species not commonly used in research have not yet been analyzed. This makes it difficult to carry out molecular studies on such animals, as the sequence of genes is the basic information used in many techniques. In this study, we attempt to design primers for a real-time PCR analysis, basing on a comparative analysis of selected gene sequences of species related to Reeves’s muntjac (Muntiacus reevesi) and by identifying highly conservative regions. Results of PCR products sequencing and their alignment with the GenBank collection show that all selected primers gave products highly similar (> 90%) to the intended target (among compared species), which led us to the conclusion that our primers may be used for further analyses of gene expression.

Cystoisospora belli (C. belli) is the only pathogenic species of the Cystoisospora genus responsible for severe diarrhea in immunocompromised patients. Most common microscopic method of diagnosis is less sensitive due to intermittent shedding of oocysts. We developed a new single-run polymerase chain reaction (PCR)–based diagnostic assay for C. belli. A new single-run PCR-based diagnostic assay was standardized for the detection of C. belli. Diagnostic reproducibility and repeatability of the PCR assay were evaluated. A cross-sectional analytical study was done on a total of 354 stool samples collected from 331 immunocompromised patients with diarrhea. All the stool samples were tested for the presence of oocysts of C. belli and were also tested by our new PCR assay for C. belli. Three of the representative PCR products were confirmed by sequencing. Fisher’s exact test was used to compare the two proportions. Microscopy detected C. belli in 11/354 (3.1%) of stool samples, and the new PCR-based assay detected C. belli in 16/354 (4.5%). The new single-run PCR-based assay detected C. belli in all the stool samples which were tested positive by microscopy and additionally detected C. belli in five stool samples. The developed PCR assay detected statistically significant proportion of C. belli (p < 0.001) as compared to microscopy. The 795 base pair PCR product from one microscopy positive stool sample and two microscopy negative stool samples were confirmed by sequencing. Our newly developed single-run PCR-based detection assay for C. belli is robust and reproducible. It may be used for molecular diagnosis of cystoisosporiasis especially in transplant, pediatrics, and human immunodeficiency virus (HIV) positive patients.

The purpose of the current study is to report and provide the first record of the macroalgal diversity observed along the underrated northern coast of the Gulf of Kachchh (GoK), India, backed by its molecular study. The biodiversity data existing for the northern belt of the GoK, encompassing the Kachchh coast, is quite flimsy in contrast to the substantial amount of seaweed diversity data obtainable for its southern counterpart. Four Chlorophytes were collected from the selected sites and subjected to molecular characterization using internal transcribed spacer (ITS) regions, (ITS1 & ITS2). The ITS1 and ITS2 regions were amplified using ITS1 and ITS4 primers. The purified PCR products were sequenced. The G + C ratio of the ITS region ranged from 40.76 to 57.34%. The purity ratio ranged from 1.68 to 2.10, while the DNA concentration varied from 30 to 168 ng/µL for 4 different species tested. The average yield of the DNA recorded for these Chlorophytes was 5.45 µg. The phylogenetic tree was created by aligning the ITS sequence using MEGA 7 software following the maximum likelihood method. The taxonomic identity of all these 4 macroalgal species was successfully determined based on PCR results. The GenBank accession numbers [(MT452260), (MT452261), (MT484071), and (MT452262)] were obtained for all the four sequences. Overall, the northern region is species-poor in comparison with the southern belt of the Gulf of Kachchh, yet it bears some economically viable macroalgal species making it an important ecological area to be studied and explored.

Objective We aimed to simplify our fetal RHD genotyping protocol by changing the method to attach Illumina’s sequencing adaptors to PCR products from the ligation-based method to a PCR-based method, and to improve its reliability and robustness by introducing unique molecular indexes, which allow us to count the numbers of DNA fragments used as PCR templates and to minimize the effects of PCR and sequencing errors. Results Both of the newly established protocols reduced time and cost compared with our conventional protocol. Removal of PCR duplicates using UMIs reduced the frequencies of erroneously mapped sequences reads likely generated by PCR and sequencing errors. The modified protocols will help us facilitate implementing fetal RHD genotyping for East Asian populations into clinical practice.

A variant of multiplex PCR on a chip with direct detection of immobilized primer elongation has been developed. Detection is performed by determining the fluorescence signal of labeled dUTPs incorporated in the growing chain. This approach creates an elongated immobilized primer with a covalently bound fluorescent label. The product of a primer elongation carries several fluorescent labels (depending on the AT composition and length of the PCR product), which allows the complete removal of all the components of the mixture after PCR and thus dramatically reduces the background signal and increases the sensitivity of analysis on hydrogel microchips. The method was successfully applied for differential detection of important bacterial pathogens of human pneumonia Staphylococcus aureus and Streptococcus pneumoniae, including testing of the one analyzed sample for the both pathogens.

Dacrycarpus imbricatus Blume is a member of the Podocarpaceae family. In Bali, D. imbricatus was found in Bukit Tapak, Tabanan Regency. This species is one of the dominant species in Bukit Tapak. This study aimed to determine the genetic variation of D. imbricatus in Bukit Tapak using molecular markers RAPD (Random Amplified Polymorphic DNA). The genetic diversity of D. imbricatus needs to be studied to obtain the information used for the conservation of this species. Leaf samples were taken from Bukit Tapak, Candikuning, Baturiti, Tabanan Regency, Bali. DNA was extracted using the CTAB method, followed by extraction using chloroform: isoamyl alcohol. DNA precipitation was carried out using ethanol. RAPD analysis was performed using polymerase chain reaction (PCR) using four primers. PCR products were visualized using agarose gel electrophoresis and ethidium bromide staining. The results showed that the amplified DNA bands ranged from 1 to 5 bands with DNA band sizes ranging from 230 bp - 422 bp. Only OPA4 and UBC106 primers can be used to detect D. imbricatus diversity based on the H, D, and R values. The detected genetic variation is low, as indicated by an average polymorphism of 32.5% and similarities between samples ranging from 0.51 to 1

Class 1 integrons are mobile genetic elements that facilitate the spread of antibiotic resistance genes among bacteria. The use of prophylactic antibiotics has resulted in the rise of antibiotic resistance genes accumulating in a wide range of settings, including poultry houses and the agricultural fields where poultry litter is applied as a fertilizer. Biochar and wood vinegar are forest products wastes that have generated increasing attention as additives to agricultural soils. The objectives of this study were to observe the prevalence of class 1 integrons in poultry litter blended with biochar and wood vinegar over time and to verify a modified class 1 integron screening assay. Poultry litter blends were sampled and screened for class 1 integrons using polymerase chain reaction, and 80 products, 79 of which showed positive, were sent for DNA sequencing. The GenBank® BLAST database was used to verify the presence of the class 1 integron-integrase gene (intI1). There was no change in prevalence over time in poultry litter blends. Out of 79 PCR products that were intI1 positive, 78 showed at least 95% sequence identity to intI1 encoding bacteria and 64 showed at least 97% sequence identity. This indicates that this method was effective for conducting baseline surveillance of class 1 integrons in poultry litter and poultry litter-blended biochar and/or wood vinegar. Most significantly, class 1 integron prevalence did not decrease over time, further supporting the recalcitrance of these elements and the need for improved monitoring systems.

Sarcocystis spp. are cysts-forming coccidian parasites which infect several animals including camels as intermediate hosts. The present study was designed to study the Sarcocystis infection in camels (Camelus dromedaries) at morphological and molecular levels. Sample of the esophageal, heart and ocular muscles tissue were collected from infected camels from El-kharga (New valley Governorate) and examined for Sarcocystis spp. using macroscopic evaluation, light microscopy (LM), transmission electron microscopy (TEM) and molecular analysis. Two species Sarcocystis cameli and Sarcocystis ippeni were recognized. Sarcocysts were thin walled with barely visible projections. Using the TEM, the two structurally distinct sarcocysts were recognized by unique villar protrusions (Vp). Sarcocysts of S. cameli had Vp of type 9j. The sarcocyst wall had upright slender Vp, up to 0.1-0.4 μm long and 0•05-0.1 μm wide. On each Vp, Rows of knob-like protrusions are present appeared to be interconnected. Sarcocystis ippeni had ‘‘type 32’’ sarcocyst wall characterized by conical Vp with an electron dense knob. The Vp were approximately 175.27-266.76 nm long, 100.45-175.98 nm wide. In each Sarcocystis, the Vp had microtubules (Mt) that originated at midpoint of the ground substance (Gs) and continued up to the tip. Molecular data revealed the amplification of partial fragments of the 18S rRNA gene (~600 bp). The digestion analysis of obtained PCR products using RFLP method utilizing (Mbo1) appeared 2 bands approximately 250, 350 bp for each Sarcocystis. Molecular analysis demonstared the ability of 18S rRNA gene for distinguishing Sarcocystis species in studied animals and to be used as molecular marker.

The aim of this study was to investigate equine piroplasms of wild horses (Equus ferus caballus) in Konya province of Turkey in November-December 2017. For this aim, blood samples were collected from 36 wild horses and examined for equine piroplasms by microscopy and multiplex PCR. Some of the PCR products from positive samples were also sequenced. Five (13.89%) out of the 36 horses were infected with either Theileria equi, Babesia caballi or both in the microscopical examination. Single infections with T. equi and B. caballi were detected in three (8.33%) and one horses (2.78%), respectively. Prevalence of T. equi, B. caballi and mix infections was determined as 50%, 38.8% and 38.8% by multiplex PCR, respectively. Multiplex PCR was found more sensitive than microscopical examination to detect the piroplasms of horses. The results of sequence analysis showed 99.25-100% and 98.23-99.59% nucleotide sequence identity to the previously reported T. equi and B. caballi 18S rRNA gene sequences, respectively. Consequently, the existence of equine piroplasmosis in wild horses was reported for the first time in Turkey, and high molecular prevalences of T. equi and B. caballi were reported with this study.

This study was carried out to estimate the genetic diversity between cultivars of alfalfa using SRAP marker system (sequence-related amplified polymorphism). We tested 25 combinations of SRAP markers and revealed that seven of them were informative for DNA polymorphism identification. These combinations generated 129 PCR products with the percentage of polymorphic bands per pair ranging from 21 to 50%. Specific amplification products were identified for 14 cultivars for genetic certification.

Single-strand conformation polymorphism (SSCP) is a reproducible and sensitive method for the detection of genetic polymorphisms and mutations in a wide range of polymerase chain reaction (PCR) products. However, the applications of this technique are largely confined to a set of cumbersome optimizations. Herein, a non-time-consuming method for PCR-SSCP that can be conducted with minimum efforts and less technical expertise is presented. The main concept of this simplified technique was based on many optimizations that were conducted to ensure the highest possible sensitivity to detect genetic polymorphism without incorporating sophisticated equipment and tedious efforts. The optimum gel concentration, temperature, and time requirements were strictly adjusted so as not to be further modified before applying this method for genotyping purposes. Furthermore, minimized silver staining steps were combined with this method to further minimize time and effort. It was confirmed that the performed adjustments were not reduced the overall sensitivity of the technique. Therefore, the suggested method can be utilized to genotype a wide range of PCR products (mainly from 200 to 600 bp) without the need for further optimizations and modifications. This study proposes a rapid SSCP protocol for genotyping PCR products using simple, low-cost, and friendly to perform recipes.

Objectives: Tuberculosis (TB) is a dangerous and fatal infection. Phospholipid genes can be involved in the pathogenesis of this disease. The aims of this study were evaluation of the plcD gene prevalence and polymorphisms in some clinical Mycobacterium tuberculosis and the position of IS6110 element in this gene. Methods: This study was conducted on 250 clinical M. tuberculosis isolates. The frequency and polymorphism of the plcD gene were detected by PCR-Restriction Fragment Length Polymorphism (RFLP). As well as, the ability and place of the IS6110 element to insert into the plcD gene were evaluated. Results: The plcD gene was present in 88 (35.2%) isolates. Among them, 73 cases (82.95%) had a normal PCR product without the IS6110 element, and the size of PCR products of plcD was 2837 bp in other positive cases, which indicated the presence of the IS6110 element. Surprisingly, the plcD gene was absent in all Beijing isolates. Conclusions: Based on the data, the plcD gene is one of the normal sites for the insertion of the IS6110 element. In addition, these findings indicated that the role of plcD in the pathogenesis of TB may be covered by other plc genes, as this gene was deleted in all Beijing isolates studied in this research.

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) system was modified and evaluated for the simultaneous detection of multiple viruses in coinfected lily plants. Four major lily viruses, namely, lily symptomless, cucumber mosaic, lily mottle, and plantago asiatica mosaic viruses, were targeted by simultaneous detection. Analysis of PCR products confirmed the amplification of virus-specific DNA fragments of the expected sizes and nucleotide sequences from the target viruses. The cDNAs from singly infected plant samples were used as positive controls, and multiple infected samples were used to develop the multiplex RT-PCR system, which was then applied to leaf samples from diverse lily species and hybrids. The modified multiplex RT-PCR was successfully validated on the simultaneous detection of these viruses.

Dermatophagoides pteronyssinus and Dermatophagoides farinae are the most common House Dust Mite (HDM) species in home environments worldwide and responsible for HDM allergy. Since the prevalence of HDM-related clinical conditions is linked to exposure to the mite itself, the detection of HDM in the human households gains importance. We aimed to develop a fast and accessible multiplex PCR to detect and distinguish two relevant HDM species in house dust. New primers were designed, and sensitivity analysis was performed. Sequencing of PCR products was also performed to confirm the method's specificity. The limit of detection of the multiplex PCR for both species was as low as 30 pg µL-1. The application of the multiplex PCR to dust samples also resulted in the identification of both species with high sensitivity. The protocol required small amount of template, reagents and a short reaction time thus presenting an alternative to classically used techniques for HDM identification. This work was supported by the Foundation for Science and Technology (FCT), through funds from the State Budget, and by the European Regional Development Fund (ERDF), under the Portugal 2020 Program, through the Regional Operational Program of the Center (Centro2020), through the Project with the Grant [UIDB/00709/2020]. Financial support was also provided by the Foundation for Science and Technology (FCT) through a PhD fellowship to A.S.O under Grant [SFRH//BD/136192/2018]. info:eu-repo/semantics/publishedVersion

The attack of WSSV in Vannamei shrimp cultivation is still common. Shrimp quality improvement can be made through selection with the help of markers (marker-assisted choice). This study aimed to evaluate the DNA fragment profile of white shrimp that was resistant to WSSV disease. The analysis was performed using the PCR-RAPD method. WSSV challenged four groups of 100 Vannamei shrimp, then DNA was extracted from live and dead shrimp. The results showed that 2 of the 17 primers tested had high potential as markers, namely OP-09 and OPD-2. PCR products with OPC-09 primers had specific DNA bands measuring about 1.2 kb in all post-challenge WSSV resistant shrimp individuals. The amplification results using OPD-02 primers showed a particular band of DNA with a length of about 1.0 kb, with 60 % of the appearance in WSSV-resistant shrimp. In contrast, the WSSV-susceptible shrimp group did not have specific DNA fragments. Thus, the two RAPD primers had a high chance of being used in the selection with the help of markers to produce WSSV resistant shrimp.

The distribution and frequency of potato leaf roll virus in the four most important potato growing regions in Serbia were studied during the seven years (2012–18). One hundred randomly collected potato tubers were sampled from each seed lot. The young leaves that developed in three weeks were sampled and tested to record infection rate. The presence of potato leaf roll virus was detected by double-antibody sandwich enzyme-linked immunosorbent assay and disease incidence was calculated using standard formula. The obtained result showed that the highest prevalence of potato leaf roll virus was detected from seed potato samples originated from the Raski region during 2018 (20.7%), while in the Moravicki region, only 2.3–11.1% of the potato leaf roll virus was detected every year. The average annual potato leaf roll virus infection was the highest in 2012 (8.4%) and 2018 (8.0%). For further confirmation of potato leaf roll virus infection, reverse transcription (RT)-PCR was performed using specific primers PLRVCPvEcoRI /PLRVCPcNcoI, designed to amplify a 650 bp fragment of the full-length coat protein gene. The PCR products derived from 26 isolates were directly sequenced using the same primer pair as in RT-PCR. The coat protein sequence analysis revealed that the Serbian potato leaf roll virus isolates showed very low nucleotide diversity (95.9–100%). They shared the highest nt identities of 98.08–99.36% with the sequences of potato leaf roll virus isolates deposited in the GenBank from other parts of the world. Phylogenetic analysis and the haplotype network of the coat protein gene sequences showed that the Serbian potato leaf roll virus isolates could be classified in two different groups indicating two possible introductions of the virus to Serbia. The results of this study confirmed the importance of potato leaf roll virus in seed potato production in Serbia. Additionally, this research highlights the need for a continuous monitoring of the potato seeds produced in Serbia as well as imported seeds for the presence of potato leaf roll virus.

The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 96 samples at a time. There is a need, however, for a flexible, platform agnostic, method that can provide multiple throughput options depending on changing requirements as the pandemic peaks and troughs. Here we present CoronaHiT, a method capable of multiplexing up to 96 small genomes on a single MinION flowcell or >384 genomes on Illumina NextSeq, using transposase mediated addition of adapters and PCR based addition of barcodes to ARTIC PCR products. We demonstrate the method by sequencing 95 and 59 SARS-CoV-2 genomes for routine and rapid outbreak response runs, respectively, on Nanopore and Illumina platforms and compare to the standard ARTIC LoCost nanopore method. Of the 154 samples sequenced using the three approaches, genomes with ≥ 90% coverage (GISAID criteria) were generated for 64.3% of samples for ARTIC LoCost, 71.4% for CoronaHiT-ONT, and 76.6% for CoronaHiT-Illumina and have almost identical clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 96 SARS-CoV-2 genomes per MinION flowcell and that Illumina sequencing can be performed on the same libraries, which will allow significantly higher throughput. CoronaHiT provides increased coverage for higher Ct samples, thereby increasing the number of high quality genomes that pass the GISAID QC threshold. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.

Genetic purity is conventionally performed through grow-out-test (GOT) with morphological characters. Simple sequence repeat (SSR) markers are independent of G X E interaction. Herewith, 16 high yielding varieties of rice were analysed using 55 SSR markers for DNA fingerprinting and identification of genetic impurities: 14 were found to be polymorphic and amplified 48 alleles with an average of 3.43 alleles per each primer pair. The number of alleles amplified ranged from 2 to 6 and the size of the PCR products amplified from these 14 primer pairs ranged from 80 to 450 bp with polymorphic information content (PIC) from 0.14 (RM 346) to 0.99 (RM 5900). PICs at 0.5 or higher are highly informative SSR markers for genetic studies and the study reported (7/14) markers to be highly informative based on PIC values. Besides, the values of effective multiplex ratio, marker index and resolving power for the selected polymorphic primer pairs disclosed that the SSR markers used in the study are highly informative and could be potentially used for distinctness, uniformity and stability (DUS) testing, genetic purity analysis and DNA fingerprinting of rice varieties. Economic analysis of grow-out-test and SSR marker based genetic purity showed that the GOT process incurs Rs. 5.8 (INR) per seed (USD 0.07), while DNA-based marker analysis incurs Rs. 53.75 (INR) or $ 0.71 (USD).

Background: Staphylococcus aureus (S. aureus) causes a difficult problem in the poultry industry because it causes diseases that are difficult to treat due to the resistance of these bacteria to antibiotics and their possession of a battery of virulence and resistance genes in addition to their ability to produce thick biofilms. Method: A cross-sectional study conducted to collect a total of 53 samples from different clinical cases in broilers during the period from August 2019 to February 2020 in Al-Najaf and Karbala cities, The clinical isolates were determined by using the conventional standard biochemical tests. All the specimens cultured on blood agar medium supplemented with 5% blood for primary isolation and selected by using selective media mannitol salt agar (MSA) for confirmation the mannitol fermentation, then subjected to gram’s staining, catalase, oxidase, and further slide coagulase test, then all S. aureus isolates tested by antibiotic susceptibility test, and screened for the presence of mecA and mecC genes using PCR for the detection of MRSA isolates, then subjected to the detection of virulence genes (pvl and eta), antibiotic resistance gene (cfr), identification of integron class 1, biofilm formation assay, the multi-druge resistance profiles (MDR) and multible antibiotics resistance (MAR) indexes were calculated. Results: the isolation rate of S. aureus from the broilers' clinical samples was 37.7%. The antibiotic susceptibility test revealed that 85% of S. aureus isolates were resistant to one or more of the antibiotic tested. All 53 isolates were assessed for the presence of mecA and mecC genes by using PCR. The mecA gene-specific PCR product was seen in 7 (35%) isolates and considered as MRSA. Among all S. aureus isolates, two isolates were positive for the eta gene, and 15 (75%) isolates harboring integron class 1, while the biofilm formation test revealed that 7 (35%) was positive biofilm producers and three of them were strong producers, consequentlly, 13 (65%) of the isolates were resisted to three or more antibiotics and considered as MDR strains. While pvl, cfr, and mecC gene were not detected among S. aureus isolates. Conclusion: the current study revealed that S. aureus possess a real threat in the poultry industry reflecting a public health problem due to the large acquisition of antibiotic resistance genes by these bacteria, the results indicated a high percentage of isolates having MDR characteristic, and two of them were resistant to all antibiotics tested. In addition to the presence of two MRSA isolates carrying the eta gene, this indicating that they are of human origin.

Purification of DNA molecules from a large number of samples is laborious, costly, time-consuming, and a high risk of contamination. S. burahol leaves contain phenolic compounds, flavonoids, and terpenoids that can interfere with DNA isolation. Direct PCR kits can detect genes without DNA extraction. The objective of study was to determine the method of gene detection of Stelechocarpus burahol using the direct PCR kit. S. burahol leaf samples came from the Bogor Botanical Gardens (two trees), Garut, Purwodadi Botanical Gardens, Kyai Langgeng Gardens, Yogyakarta Palace, Turi Sleman, Wanagama, Karanganyar, and South Kalimantan. Each leaf sample of 0.1 mg was dissolved into 1.25% w / v SDS (Sodium Dodecyl sulfate) 50 ul solution, incubated at 95 o C for 5 minutes, and vortexed for 2 seconds. Primers used for the trials were ITS 1F primers and 4R primers. Pre-denaturation of 95 o C for 7 minutes, denaturation of 95 o C for 1 minute, annealing 55 o C for 1 minute, extension at 72 o C for 1 minute, extension at 72 o C for 1 minute, and 40 cyclus. PCR product samples of 40 - 50 μl that showed positive results were detected by electrophoresis. The results of S. burahol DNA amplification measuring ± 750 bp from ten samples of S. burahol. Direct PCR kits can be used for S. burahol gene detection, time and energy efficient, only requires a small amount of tissue, and reduces contamination due to DNA extraction. Direct PCR kits can be an effective method that can be utilized to detect target genes for large populations

Knowing the regional lineages/sublineages of HPV 31 and 45 would be of great importance for further evolutionary, epidemiological, and biological analysis. In this regard, to characterize more common lineages and sublineages of HPV 31 and 45, the sequence variations of E6 gene were investigated in normal, premalignant, and malignant samples collected from the cervix in Iran. In total, 54 HPV 31- and 24 HPV 45-positive samples were analyzed by hemi-nested PCR and nested-PCR, respectively. All PCR products were subjected to direct sequencing analysis. The results indicated that all three lineages A, B, and C were detected in HPV 31-positive samples; among which HPV 31 lineage A was dominant as it was found in 66.7% of all samples. HPV 31 lineages B and C were identified in 5.5% and 27.8% of samples, respectively. In HPV 45-infected samples, lineage B comprised of 62.5% of all samples and the remaining 37.5% was belonged to lineage A. In conclusion, our findings showed that lineage A of HPV 31 was predominant in Iran. Lineage B of HPV 45 was also dominant among Iranian women. However, further studies with larger sample size should be addressed to estimate the pathogenicity risk of HPV 31 or HPV 45 lineages/sublineages in development of cervical cancer among Iranian women. This article is protected by copyright. All rights reserved.

This study is carried out to investigate the prevalence of Coxiella burnetii (C. burnetii) infections in cattle using an enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assay targeting IS1111A transposase gene. A total of 130 lactating cows were randomly selected from different areas in Wasit province, Iraq and subjected to blood and milk sampling during the period extended between November 2018 and May 2019. ELISA and PCR tests revealed that 16.15% and 10% of the animals studied were respectively positive. Significant correlations (P

This study was aimed to identify dermatophytic selective isolate using PCR technique as a rapid molecular assay. The results of this study showed among 60 samples of patients suffering from ringworm disease. forty isolates (66%) were Trichophyton mentagrophytes which diagnosed as dermatophytosis according to morphological and cultural methods. In order to investigate the ability of isolates to keratin analyses using solid medium supplemented with keratin azure , the results revealed that 20 isolates appeared best ability to keratin analysis and nine isolates had best ability for keratinase production in submerged culture. According to this results T. mantagrophytes (K3) (had higher activity for keratinase) was chosen for molecular identification. The results of PCR revealed that primer for18S rRNA gene of T. mentagrophytes K1 isolate and specific primer for subtilisin like protease gene were amplified and appeared as single DNA band with a molecular base of 690 bp and 623bp respectively .The blast result of sample sequences of amplified fragment revealed that the isolate were 100% identical to reference sequence of T.mentagrophytes var. interdigtal and depending on data base in NCBI The result of PCR product for enzyme showed new type named GBF60362 (402) subtilisin-like protease related to T. mentagrophytes 1354684064 BFBSOLP00892.

We have developed a polymerase chain reaction (PCR) assay to facilitate detection of the major disease-associated serotypes of fowl adenovirus (FAdV) including serotypes 1, 2, 4, 8a and 8b; primers were designed based on serotype-specific sequences of the hexon gene. We tested field isolates from chickens diagnosed with inclusion body hepatitis, gizzard erosion and hydropericardium syndrome together with reference FAdV strains characterized in Japan. We found that the primers were serotype specific; appropriate amplification of serotype-specific hexon genes was confirmed by sequence analysis of the PCR products. This PCR assay will be useful for detection of FAdV and for differentiation between disease-associated serotypes.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435 bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448 bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448 bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210 bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-020-04849-w) contains supplementary material, which is available to authorized users.

The successful production of recombinant protein for biochemical, biophysical and structural biological studies critically depends on the correct expression organism. Currently the most commonly used expression organisms for structural studies are E. coli (ca. 70% of all PDB structures) and the baculovirus/ insect cell expression system (ca. 5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows the simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N- and C-terminal affinity, solublisation and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes.

Analysis of wolves dietary is a currently important theme because of the discussion about wolves preying on livestock as sheep or goats. We developed molecular markers to especially amplify the DNA of the prey out of wolf scat. For this purpose, we used the mitochondrial D-Loop using public available sequences for wolf and seven potential prey species (even-toed ungulates). We developed special primers amplifying either the wolves DNA or the prey DNA. In a fragment of 223-225 basepairs (bp) length we identified 21 SNPs, two 1-bp indels and one 3-bp indel, and three microsatellites to separate seven prey species from each other. Validation of the markers was performed by sequencing the PCR products of 12 fresh prey tissues and 20 wolf scat samples using the different primer pairs.

The production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method’s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = −3.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food.

Three new species-specific primer sets were designed to detect C. truncatum, C. gloeosporioides species complex, and C. scovillei, which were reported as the cause of chili anthracnose disease. These new primers were designed based on the sequence data of the nuclear ribosomal DNA (rDNA) region to amplify and identify short size target DNA (

The aim of the study was to analyze the associations between SNPs in the creatine kinase gene (CKM) and racing performance of homing pigeons. A 468-base pairs fragment of the CKM gene (a part of exon 11 and 3'UTR) was amplified. Five PCR products were sequenced. Two substitutions in the amplified region were observed (g.588268 T>C and g.588285 T>C). PCR-RFLP method was used to genotype 123 racing pigeons (CKM/Hpy188I and CKM/BsrDI) for the further association analysis. The frequencies of genotypes analyzed were: CKM/Hpy188ICC – 0.008, CKM/Hpy188ICT – 0.057, CKM/Hpy188ITT – 0.935 and CKM/BsrDICC– 0.008, CKM/BsrDICT– 0.016, CKM/BsrDITT– 0.976. The effect of one SNP studied on racing performance of pigeons was statistically significant. The largest difference in ace points (APs) was found for CKM/BsrDI (g.588285 T>C). Individuals with the CC and CT genotypes had lower racing performance than TT ones. The differences between CC and TT pigeons were statistically significant (p = 0.0391). Thus, racing pigeons with the most common genotypes (TT) had better racing performance than individuals with minor allele in genotype. However, the observation of an individual with CC genotype in the CKM/Hpy188I with the highest APs in two seasons studied complicates the final conclusion. Based on the obtained results, it may be concluded that two SNPs in the CKM detected in this study were not the major determinant of racing performance in sport pigeons.

DNA barcoding is a valuable tool to support species identification with broad applications from traditional taxonomy, ecology, forensics, food analysis, and environmental science. We introduce Microfluidic Enrichment Barcoding (MEBarcoding) for plant DNA Barcoding, a cost-effective method for high-throughput DNA barcoding. MEBarcoding uses the Fluidigm Access Array to simultaneously amplify targeted regions for 48 DNA samples and hundreds of PCR primer pairs (producing up to 23,040 PCR products) during a single thermal cycling protocol. As a proof of concept, we developed a microfluidic PCR workflow using the Fluidigm Access Array and Illumina MiSeq. We tested 96 samples for each of the four primary DNA barcode loci in plants: rbcL, matK, trnH-psbA, and ITS. This workflow was used to build a reference library for 78 families and 96 genera from all major plant lineages – many currently lacking in public databases. Our results show that this technique is an efficient alternative to traditional PCR and Sanger sequencing to generate large amounts of plant DNA barcodes and build more comprehensive barcode databases.

Giardia intestinalis is a flagellated protozoan that lives and proliferates in the small intestine of the host causing giardiasis. The route of transmission is the fecal–oral route, either directly or indirectly. Limited genetic information on G. intestinalis is known in Algeria. This study aimed to estimate the prevalence of G. intestinalis assemblages in the city of Djelfa. A total of 355 fecal samples were collected from symptomatic and asymptomatic school children aged ranged between 6 and 11 years old. Genotyping was done to the Giardia positive samples (n = 30) targeting the beta-giardin gene by applying PCR/RFLP assay. Our data showed that most of the cases were asymptomatic (56.7%). Co-infection with other intestinal parasites was found in 16.6% of cases. We obtained 28/30 positive PCR products while two samples only showed false-negative results, and only 20 samples have shown strong PCR products suitable for RFLP analysis. Assemblage A (70%) was more prevalent than assemblage B (30%) and was more expressed by signs than assemblage B. Moreover, only assemblage A was associated with close contacts with domestic animals and birds. In conclusion, this study gave the first molecular data on G. intestinalis isolates in the city of Djelfa. Further expanded studies using more genes and covering other cities in Algeria are mostly needed.

In November 2016, leaf curling and yellowing were observed in common bean plants on Ishigaki Island, Okinawa Prefecture, Japan. Those symptoms suggested a virus-like disease. PCR using specific primers for ageratum yellow vein virus (AYVV) yielded PCR products of the expected size from diseased plants. The complete nucleotide sequence of the DNA-A shared 99.9% identity with that of the AYVV-Ishigaki isolate. Further, the virus was transmitted by Bemisia tabaci Middle East-Asia Minor 1 and Mediterranean. This is the first report of the natural occurrence of AYVV in common bean plants.

This study is aimed to analyze the comparative quality of PMSA2 gene amplicon product stability from two different specimen sources, spot specimens of dried blood and venous blood, as well as selecting the best storage method for specimens of blood samples. This research uses descriptive laboratory research methods. The research began with the process of sample preparation for dried blood spot and venous blood, each using Whatman 903 paper and vacuum tubes containing EDTA, isolating genomic DNA using KIT Zymo Research, amplification of PMSA-2 genes with PCR, detection of PCR products through electrophoresis, measurement of DNA concentration and absorbance, and data analysis. The results of this study are expected to be a source of information about the advantages of two specimen storage methods for clinical blood samples, as well as providing a clear description of the quality of each specimen storage method based on the quality of its amplicon products. The results showed that a total of ten medical samples of dried blood spot and ten venous blood were isolated from the genomic DNA of ten and nine, respectively. PMSA2 gene amplicons detected were seven in venous blood and six in dried blood spot. Venous blood specimens have sensitivity in detecting PMSA genes in samples with the highest value of 554 ng / μL and purity of 2,007 (WB7), and concentration of 550.2 and highest purity of 2,076 (WB10). Venous blood storage techniques using categorized vacuum tubes are effective in the detection of PMSA2 genes and have time efficiency in the process. From these results it was also concluded that the comparison analysis of amplicon products between venous blood specimens was better, more stable and efficient than dry blood spot specimens, thus recommending storage of venous blood specimens using vacuum tubes as the best storage method of blood sample specimens.

The wood adulteration is a common problem and under-studied aspect in the timber industry of Sri Lanka. Hence we conducted a survey to assess the status of timber adulteration and check the applicability of morphometric parameters and DNA barcoding to detect the adulterated timber sources. We interviewed the stakeholders of the timber industry to collect information regarding timber adulterations. We measured the morphometric parameters; wood density and sizes of the xylem elements of the standard and adulterant species. For DNA barcoding, DNA was extracted from the wood of the selected standard and adulterant species and subjected to PCR using the markers, matK-trnT and atpB-rbcL. The PCR products were subjected to DNA sequencing. According to the survey, 92.5% of patrons, 73.7% of manufacturers and 96.7% of carpenters said timber adulteration is taking place in the country. The respondents said that the standard timber species; Tectona grandis, Artocarpus heterophyllus, and Swietenia macrophylla, profoundly undergo adulteration in Sri Lanka. The morphometric parameters did not discriminate the adulterant species from the standard species. The DNA barcodes matK-trnT and atpB-rbcL provided unique polymorphic DNA sequences with specific lengths for each species permitting the precise establishment of species identity and enabling the accurate detection of timber adulterations.