One-step Polymerase Chain Reaction Research Articles

A small review on polymerase chain reaction for the detection of Salmonella species

Salmonella identification from blood samples is crucial for rapid detection and efficient medication of typhoid and paratyphoid fever. Because of its remarkable sensitivity and specificity, polymerase chain reaction (PCR) is a broadly applied technology. The goal of this analysis of 16 papers concentrating on PCR-based Salmonella species identification in blood samples is to identify the most common and successful PCR techniques. The review covers a variety of PCR methods, such as one-step differential detection PCR, nested PCR, multiplex PCR, and real-time PCR. The effectiveness of many PCR primers, including those for the flagellin gene, hilA gene, invA gene, and iroB gene, in detecting Salmonella was examined. The examined studies consistently showed that the PCR techniques used had good sensitivity (95%–100%) and specificity (97%–100%). In addition, PCR was effectively used by the researchers to identify particular species of Salmonella serovars, which comprise Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis. Notably, multiplex PCR became a useful technique for detecting many Salmonella serovars at the same time. The use of PCR in identifying antibiotic resistance in Salmonella isolates is also emphasized in the review. The collective results highlight the remarkable specificity and sensitivity of PCR-based techniques for Salmonella species identification from blood samples. Of them, real-time PCR and multiplex PCR are the most widely used because of their increased efficiency, sensitivity, and specificity.

The inverse association between the apolipoprotein E ε4 allele and C-reactive protein levels is stronger in persons with obesity and diabetes.

C-reactive protein (CRP) is lower in patients who carry the apolipoprotein E epsilon 4 allele variant (APOEε4) of the APOE gene. This could however be explained by other factors observed in APOEε4 carriers, such as lower body mass index (BMI), possibly less diabetes and more use of statins, all associated with CRP concentrations. To assess the association between CRP and APOEε4 stratified by BMI, statin use and diabetes. We included 2700 community-dwelling older adults from the Hordaland health study with genotyping of the APOE gene by a one-step polymerase chain reaction and CRP measured using immuno-MALDI-TOF MS. Differences in CRP concentrations by APOE (ε4 vs no ε4) were assessed using the Mann-Whitney U tests, also stratified by statin use, diabetes and BMI categories. Finally, we performed linear regression with log (CRP) as the outcome and APOEε4 together with statin use, diabetes, BMI and their respective interactions. CRP was higher in APOEε4 carriers irrespective of BMI, diabetes and statin use. In APOEε4 non-carriers, CRP was elevated with diabetes and obesity as expected. However, this was attenuated or even reversed in APOEε4 carriers. Such differences were not observed for statin use. Statin use, obesity or diabetes did not confound the known association between the APOEε4 allele and lower CRP. Our data suggest that CRP is less responsive to inflammatory cues involved in diabetes and obesity in APOEε4 carriers. Epidemiological studies should take note of these relationships, as CRP, APOEε4, diabetes and obesity are both linked to neurodegenerative and cardiovascular disease.

Development of a Novel Real-Time Quantitative PCR Method for Detection of Ilyonectria robusta, the Predominant Species Causing Ginseng Rusty Root Rot.

Rusty root rot is the most destructive soilborne disease of ginseng caused by pathogenic Ilyonectria spp., predominantly Ilyonectria robusta, in China. However, there remains no effective strategy to control the disease. Current control of the disease requires that soil and ginseng seeds and seedlings infected with I. robusta are avoided during planting. Therefore, rapid and accurate detection of I. robusta would be indispensable in disease control programs. A one-step polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) assay was developed to detect I. robusta in ginseng seeds, roots, and soil. The species-specific primers HIS H3-F and HIS H3-R, designed based on a partial histone gene sequence of I. robusta, yielded a 268-bp product using the optimized PCR and qPCR protocol. DNA of I. robusta was detected by qPCR in all diseased soil and ginseng roots and seeds resulting from artificial inoculation and sampled from natural fields. I. robusta was detected at an abundance of 1.42 fg/μl at 12 h postinoculation and 191.31 fg/μl at 7 days postinoculation in ginseng roots that showed disease symptoms. In naturally infected soil sampled from ginseng fields, pathogen abundances ranging from 13.23 to 503.39 fg/μl were detected, which were 2.04 to 11.01 times higher than those in ginseng roots. The pathogen was first detected and was more abundant on the surface of the ginseng seed coat compared with that in the seed kernel. This study provides a high-efficiency detection technique for early diagnosis of I. robusta and real-time disease prevention and control strategies.

Comparative evaluation of some techniques used for the detection of rabies virus.

Neurotropic viruses in the family Rhabdoviridae, genus Lyssavirus, are what cause rabies, an acute, progressive, and highly lethal encephalomyelitis. Evaluation of the used diagnostic techniques to determine the most simple; rapid and accurate test for rabies virus (RABV) recognition in different specimens aiming to reach a rapid diagnosis as a step aid in the disease control and to prevent or even minimize the suspected hazard. The used techniques included an infection trial of Swiss mice with the mice-adapted challenge rabies virus followed by the detection of the virus in the infected mices' brains. Virus detection was carried out through the application BHK21 cell line infection; fluorescent antibody technique; latex agglutination test (LAT); direct enzyme-linked immunosorbent assay (ELISA); rabies antigen detection kit ELISA; conventional polymerase chain reaction (PCR). It was found that virus inoculation in mice and BHK21 cell lines needs 5-7 days with positivity of 90% and 100%, respectively. Rapid antigen kit was able to detect rabies antigen in mice brains suspension and BHK21 infected fluid within 3-5 minutes with percentages of 60% and 55.5%, respectively. In 1-1.5 hours, the direct fluorescent antibody method (DFAT) detected 90% and 100% of the rabies antigen in BHK21 cell line infection and brain impressions, respectively. Latex agglutination showed clear results with 88.8% with BHK21 infected fluid within 3-5 minutes while it did not carry out on brain emulsions to prevent falsely positive results brought on by the presence of tissue fragments. Conventional one-step PCR revealed 100% positivity with either brain or cell culture preparations within 2 days. Direct ELISA showed 88.8% positivity with BHK21 infected fluid with 1 day of work. Mice inoculation test, cell culture infection; DFAT and PCR are the most accurate techniques for the detection of RABV with a positivity of 90%-100% followed by LAT and ELISA with a positivity of 88.8%, and lastly, rabies antigen ELISA kit (RAK) with a positivity of 55.5%-60% taking in consideration the required time for each. In addition, the positivity % of the applied tests revealed their sensitivity and specificity.

A one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing dollar spot on turfgrass.

Dollar spot (DS) is one of the most destructive and economically important diseases of cool- and warm-season turfgrasses worldwide. A total of six species causing DS disease in the genus Clarireedia have been described, and four of them have been reported to be distributed countrywide in China. Identification of different species of Clarireedia is a prerequisite for the effective management of DS disease. Here we report a novel polymerase chain reaction (PCR)-based method for the detection and differentiation of the four species of Clarireedia associated with DS on turfgrass in China: C.jacksonii, C.paspali, C.monteithiana and C.hainanense. Species-specific genes were identified for each species by comparative genomics analysis. Four primer pairs were designed and mixed to amplify species-specific PCR fragments with differential sizes for the four species of Clarireedia in a single multiplex PCR assay. No PCR products were generated from the DNA templates of other common fungal pathogens associated with multiple turfgrass diseases. The multiplex PCR method developed can be used for the rapid and accurate detection and differentiation of the four species of Clarireedia from pure cultures as well as from infected turfgrass blades with DS symptoms. The study developed a one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing DS on turfgrass in China, which will have important implications for DS management in China and worldwide. © 2022 Society of Chemical Industry.

A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae.

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

Investigation of SARS-CoV-2 on Ocular Surface of Coronavirus Disease 2019 Patients Using One-Step Reverse-Transcription Droplet Digital PCR.

PurposeThis study detects SARS-CoV-2 in the ocular surface through one-step reverse-transcription droplet digital PCR (one-step RT-ddPCR) and evaluates the possibility of the ocular surface as a possible transmission route.MethodsA single-center prospective observational study was designed to investigate the viral loads in ocular surface. Specimens including the conjunctival swabs, nasopharyngeal swabs and blood were synchronously collected at a single time point for all COVID-19 patients. SARS-CoV-2 loads in nasopharyngeal swabs were tested by real-time polymerase chain reaction (PCR); the blood samples and conjunctival swabs were tested by real-time PCR and one-step RT-ddPCR.ResultsSixty-eight COVID-19 patients confirmed by nasopharyngeal real-time PCR were recruited. In the single time point test, 40 cases showed positive SARS-CoV-2 detection in either the blood, tears, or nasopharynx, of which four cases were triple-positive, 10 were dual-positive, and 26 were single-positive. The positive rate of nasopharyngeal swab real-time PCR test was 22.1% (15/68). The positive rate of blood and conjunctival swabs by one-step RT-ddPCR was 38.2% (26/68) and 25% (17/68), respectively, whereas real-time PCR was all negative. Positive conjunctival swabs were significantly correlated with positive nasopharyngeal swabs (P = 0.028). The sampling lags from illness onset to sampling day in 3 out of 4 triple-positive patients and in 9 out of 10 dual-positive patients were respectively less than 9 days and less than 20 days.ConclusionOur results indicate that the positive rate of SARS-CoV-2 on the ocular surface is much higher than expected. Transmission possibility through the ocular surface may be greatly underestimated.

Epidemiologic Features and Influencing Factors of Norovirus Outbreaks in the City of Wuxi, China from 2014 to 2018.

ABSTRACT.The study investigated the genotypic changes and epidemiologic features of norovirus outbreaks and factors influencing the attack rate and outbreak duration in Wuxi from 2014 to 2018. Norovirus outbreaks, monitored through surveillance system, were investigated. The norovirus-positive specimens from outbreaks were collected and genotyped using a dual polymerase-capsid genotyping protocol based on a one-step polymerase chain reaction (PCR) amplicon. The genotypes were analyzed by Norovirus Typing Tool Version 2.0. A total of 74 norovirus outbreaks were reported in Wuxi from 2014 to 2018. Most (93.2%) norovirus outbreaks were caused by GII genotypes. The predominant norovirus genotypes in outbreaks have changed from GII.17 (20.3%) in 2014–2015 to GII.P16/GII.2 (40.5%) in 2017–2018. GII.P16/GII.2 in 2017–2018 season were more prevalent than GII.17 in 2014–2015 season (χ2 = 4.741, P = 0.029). 56.7% of the outbreaks occurred in primary schools. The re-outbreak rate was 16.2%. 66.7% of re-outbreaks were caused by norovirus variants different from previous genotypes. Outbreaks in nonprimary school settings (odds ratio [OR]: 4.007; 95% CI: 1.247–12.876) and those leading to temporary school or institution closure (OR: 20.510; 95% CI: 1.806–232.937) were reported with a higher attack rate. The outbreaks in primary schools (OR: 4.248; 95% CI: 1.211–14.903), re-outbreaks (OR: 6.433; 95% CI: 1.103–37.534) and longer report timing (OR: 8.380; 95% CI: 2.259–31.089) declared a significantly longer duration. It is of great importance that the monitoring of norovirus outbreaks for the emergence of novel strains, along with responsive prevention and control intervention should be strengthened in adults and school-age population, especially in primary students and preschool children.

One-Step Polymerase Chain Reaction-Free Nanowire-Based Plasma Cell-Free DNA Assay to Detect EML4-ALK Fusion and to Monitor Resistance in Lung Cancer.

BackgroundNext‐generation sequencing has mostly been used for genotyping cell‐free DNA (cfDNA) in plasma. However, this assay has several clinical limitations. We evaluated the clinical utility of a novel polymerase chain reaction–free nanowire (NW)‐based plasma cfDNA assay for detecting ALK fusion and mutations.Patients, Materials, and MethodsWe consecutively enrolled 99 patients with advanced non‐small cell lung cancer undergoing a fluorescence in situ hybridization (FISH) test for ALK fusion; ALK‐positive (n = 36). The NW‐based assay was performed using 50–100 μL of plasma collected at pretreatment and every 8 weeks during ALK inhibitor treatment.ResultsThere was high concordance between the NW‐based assay and the FISH test for identification of ALK fusion (94.9% with a kappa coefficient value of 0.892, 95% confidence interval [CI], 0.799–0.984). There was no difference in the response rate to the first anaplastic lymphoma kinase inhibitor between the ALK‐positive patients identified by the NW‐based assay and by the FISH test (73.5% vs. 72.2%, p = .931). In the ALK variant analysis, variants 1 and 3 subgroups were detected in 27 (75.0%) and 8 (22.2%) patients, respectively. Among 24 patients treated with crizotinib, variant 3 subgroup was associated with worse median overall survival than variant 1 subgroup (36.5 months; 95% CI, 0.09–87.6 vs. 19.8 months; 95% CI, 9.9–not reached, p = .004]. A serial assessment identified that ALK L1196M resistance mutation emerged before radiologic progression during crizotinib treatment.ConclusionThe newly developed simple NW‐based cfDNA assay may be clinically applicable for rapid diagnosis of ALK fusion with its variant forms and early detection of resistance.Implications for PracticeThe authors developed a novel one‐step polymerase chain reaction–free nanowire (NW)‐based plasma cell‐free DNA (cfDNA) assay. This study evaluated the clinical utility of this novel method for the diagnosis of EML4‐ALK fusion in advanced non‐small cell lung cancer (NSCLC). The NW‐based assay and FISH test showed high concordance rate in 99 patients with advanced NSCLC. Serial cfDNA assessment demonstrated this method provided early detection of resistance before radiologic progression during crizotinib treatment. Taken together, plasma cfDNA genotyping by the NW‐based cfDNA assay may be useful for the rapid diagnosis of ALK fusion, classifying variants, and early detection of resistance.

Development of a Quantitative One-Step RT-PCR Method for the Detection of Sabin 2 Virus Contamination in a Novel Oral Poliovirus Vaccine Type 2.

To control circulating vaccine-derived type 2 poliovirus outbreaks, a more genetically stable novel Oral Poliovirus Vaccine type 2 (nOPV2) was developed by targeted modifications of Sabin 2 genome. Since the use of OPV2 made of Sabin 2 strain has been stopped, it is important to exclude the possibility that batches of nOPV2 are contaminated with Sabin 2 virus. Here, we report the development of a simple quantitative one-step reverse-transcription polymerase chain reaction assay for the detection and quantitation of Sabin 2 virus in the presence of overwhelming amounts of nOPV2 strain. The method is specific and linear within 8 log10 range even in the presence of relevant amounts of nOPV2 virus. It is sensitive, with a lower limit of detection of 0.2 CCID50/mL (an equivalent of 198 genome copies per mL), and generates reproducible results. This assay can be used for quality control and lot release of the nOPV2.

A simple one-step PCR assay for SNP detection.

Polymerase Chain Reaction (PCR) is a powerful tool to detect natural variation or experimentally introduced variation in research and clinical settings and a widely-used method for genotyping. Single nucleotide polymorphisms (SNP) detection is challenging by PCR as the variant and wild type alleles differ by only one nucleotide. Traditional methods to detect SNPs, including Sanger sequencing and commercial kits, are usually time-consuming. Here we describe a simple primer design strategy that enables specific variant detection through regular one-step PCR. The strategy employs the differential efficiency of genomic PCR using a primer that has a single mismatch with the chromosome that contains the SNP to be detected (typically the variant allele) versus two mismatches with the corresponding alternative allele (typically the wild type allele). To date, we have successfully employed this approach to detect more than 20 SNPs. The simplicity and robustness of the approach allows rapid application to legacy mutations as well as newly discovered or generated SNPs.

Human Papillomavirus Status of Head and Neck Squamous Cell Carcinoma Patients and its Association with Expression of PIK3CA and CRNDE

Background: Human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) have distinctive molecular features. We conducted the present work to evaluate the HPV status of HNSCC patients and its association with expression of PIK3CA and CRNDE genes. Method: In the present case-control study, 50 fresh frozen tissue samples of HNSCC patients were collected from the patients referred to hospital for tumor removal. HPV typing was performed on DNA samples using a one-step polymerase chain reaction (PCR) followed by Reverse Line Blot method. Relative expression of PIK3CA and CRNDE genes was evaluated utilizing a real-time PCR method. Results: Out of 50 patients, 14 (28%) were HPV positive and the most prevalent type was HPV 16. Both PIK3CA and CRNDE genes were upregulated in tumoral tissues compared to adjacent non-cancerous tissues (ANCTs) (P=0.0322 and 0.0005, respectively). Based on the area under curve (AUC) values, the diagnostic power of CRNDE (AUC= 0.676) was higher than that of PIK3CA (AUC= 0.604). Finally, the expression level of PIK3CA was significantly associated with HPV+ HNSCC (P=0.01). Conclusion: We showed that the prevalence of HPV in HNSCC was within our local prevalence range. Moreover, the association of PIK3CA overexpression with HPV status implied distinctive molecular characteristics of HPV(+) HNSCC.

Accomplishment of one-step specific PCR and evaluated SELEX process by a dual-microfluidic amplified system.

One of the main obstacles for systematic evolution of ligands by exponential enrichment (SELEX) failure is the generation of a non-specific product, as selection-inherent amplification procedures tend to form by-products, which prevents the enrichment of target-binding aptamers. Herein, we reported a dual-microfluidic amplified system (dual-MAS) based on the real-time polymerase chain reaction (PCR) detection chip and the large volume PCR chip for one-step specific PCR and for evaluating the SELEX process. First, it is a simple method to accomplish analytical PCR and amplification PCR in one step, and the optimal number of cycles for generating the specific PCR product is the cycles when the slope of the linear amplification period of the real-time PCR curve begins to decrease. Second, the time used by the dual-MAS for generating a specific PCR product is reduced to 30 min, and the multi-functional dual-MAS can simultaneously evaluate the SELEX process by providing important information on the amounts of enriched sequences and the library diversity in every round of SELEX. In addition, pollution contamination and fragment loss can be significantly avoided in the closed chip. Last, the specific PCR product, the amounts of enriched sequences, and the library diversity can be obtained for every single SELEX in just 30 min. Compared with current methods, this system can reduce the time for generating a specific PCR product and SELEX, and it is easier to choose the optimal number of cycles for a specific PCR product. In a word, it is a sensitive, simple, and rapid strategy to improve the specificity of the PCR product and make the process of SELEX in a controlled way.

RNase in the saliva can affect the detection of severe acute respiratory syndrome coronavirus 2 by real-time one-step polymerase chain reaction using saliva samples

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded RNA virus that causes coronavirus disease 2019, which spread worldwide immediately after the first patient infected with this virus was discovered in Wuhan, China, in December 2019. Currently, polymerase chain reaction (PCR) specimens for the detection of SARS-CoV-2 include saliva, nasopharyngeal swabs, and lower respiratory tract-derived materials such as sputum. Initially, nasopharyngeal swab specimens were applied mainly to the PCR detection of SARS-CoV-2. There was a risk of infection to healthcare workers due to coughing or sneezing by the subjects at the time of sample collection. In contrast, saliva specimens have a low risk of droplet infection and are easy to collect, and their application to PCR testing has been promoted. In this study, we have determined the detection limit of SARS-CoV-2 in saliva samples and examined the effects of storage temperature and storage time of saliva samples on the PCR detection results. As a result, 5 × 103 copies of SARS-CoV-2 could be detected in 1 mL phosphate-buffered saline, whereas 5 × 104 copies of SARS-CoV-2 were needed in 1 mL saliva to detect the virus by real-time one-step PCR. Interestingly, SARS-CoV-2 (5 × 103 copies/mL) could be detected in saliva supplemented with an RNase inhibitor. Concerning the saliva samples supplemented with an RNase inhibitor, the optimal temperature for sample storage was −20 °C, and PCR detection was maintained within 48 h without problems under these conditions. These finding suggest that RNase in the saliva can affect the detection of SARS-CoV-2 by PCR using saliva samples.

KHV Detection Based on Thermal Lysis - Nested PCR (Polymerase Chain Reaction)

In the case of common carp (Cyprinus carpio) and goldfish (Carrasius auratus) that were lightly infected with Koi Herpes Virus (KHV), the use of the one-step PCR method was insensitive to detect the presence of KHV DNA. The nested PCR method has been developed for a more sensitive detection system for KHV infection. This study aims to test the sensitivity of one-step and nested PCR methods in detecting the KHV virus in lightly infected common carp to prevent early disease attacks. DNA extraction of the test samples used thermal lysis and PBS (Phosphate Buffered Saline). DNA isolate as a KHV template was used to amplify KHV sequences using KHV-TKf: 5'-GGGTTACCTGTACGAG-3 'and KHV-TKr: 5'-CACCCAGTAGATTATGC-3' primers in the one-step PCR method. The nested PCR method uses two pairs of primers, namely the KHV-TKf: 5'-GACACCACATCTGCAAGGAG-3' and KHV-TKr: 5'-GACACATGTTACAATGGTGGC-3') and the CEFAS primer pair (5'-CGTCGTGAGGAATACGACG-3' and 5'- ACCGTACAGCTCGTACTGG-3') for amplification of KHV sequences. The one-step PCR method was insensitive for detection of KHV in common carp and goldfish infected with viruses without clinical symptoms, and was sensitive enough to detect viruses with mild symptoms with a DNA fragment size of 409 bp using TK primers. The nested PCR method was more sensitive in detecting KHV DNA in fish without clinical symptoms with a DNA fragment size of 348 bp using CEFAS primers.

English

Zika, yellow fever and dengue viruses are three important flaviviruses that are epidemic and endemic in Central Africa, Middle and South America, Southeast Asia and Pacific Islands. Since these three viral diseases present with similar symptoms and there is a possibility of multiple flaviviruses co-infection, it is important to develop an effective and quick diagnostic tool. Here, we designed and validated a one-step triplex real time reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probes targeting NS5 region for detecting zika virus (ZIKV), dengue virus-4 (DENV-4) and yellow fever virus (YFV). The assay was highly specific for the target viral RNA corresponding to ZIKV, DENV-4 and YFV strains. The sensitivity of the assay was compared to one-step, RT-PCR, it showed a 100-fold higher sensitivity than RT-PCR with ZIKV and DENV and 10-fold higher sensitivity with YFV. In conclusion, the one-step, triplex, TaqMan, real time RT-PCR assay identified and distinguished ZIKV, DENV-4 and YFV by targeting single gene. Key words: Zika, yellow fever, dengue, multiplex real time reverse transcription-polymerase chain reaction (RT-PCR).

A multiplex PCR method for rapid identification of commercially important seaweeds Kappaphycus alvarezii, Kappaphycus striatus and Eucheuma denticulatum (Rhodophyta, Solieriaceae)

Taxonomic clarification of Kappaphycus alvarezii, Kappaphycus striatus and Eucheuma denticulatum based on standard DNA barcodes has proven to be useful as the phenotypic plasticity of the species can confound traditional taxonomic approaches. Molecular characterization of each isolate is time-consuming and a painstaking task as multiple runs of PCR amplification needs to be carried out and the identity of the isolates is only known after sequencing of the PCR amplicons. In this study, we have developed a simple, efficient and reproducible one-step polymerase chain reaction (PCR) based method for the identification of the three eucheumatoid species, K. alvarezii, K. striatus and E. denticulatum. Based on the nuclear ribosomal internal transcribed spacer (ITS) sequence, all the available genetic polymorphisms were reviewed and three primers were designed. The combination of these primers in the same PCR reaction results in a specific amplicon of distinct size ranging from approximately 140-400 bp to be produced for each of the three red algal species. The proposed method is found to be an effective and practical tool to aid in the selection of the desired seaweed seedlings even before they are cultivated and also the identification of commercial cultivars accurately.

High sensitivity of one-step real-time reverse transcription quantitative PCR to detect low virus titers in large mosquito pools

BackgroundMosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases primarily relies on the management of the vector. Traditional control methods are insufficient to control mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males with the aim to induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as the released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined.MethodsWe evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile virus (WNV) and Zika virus (ZIKV).ResultsCHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1 and 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed the prediction of the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection.ConclusionsMosquito-borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have a SIT component by reducing the time and the manpower needed to conduct this quality control process.

Group A Rotavirus G1P[6] Associated Fatalities in Diarrheic Nigerian Infants, Possible Impact of Enterovirus Environmental Enteric Dysfunction, and Implications for Rota-Vaccinology

AbstractGroup A rotavirus (RVA) diarrhea disease and mortality are yet unabated, particularly in developing countries. As global knowledge of specific strains associated with infant mortality is crucial for successful vaccination efforts, candidate RVA strains detected in mortality and fatal cases of severely diarrheic hospitalized infants in Akure, Nigeria were investigated.Fecal samples from comatose patients were tested for RVAs, other diarrhea viruses, and enteric bacterial pathogens. Genomic dsRNA was extracted from 10% rotavirus positive stool suspension, the VP4 and VP7 genes were reverse transcribed and amplified by one-step reverse transcription polymerase chain reaction (PCR) and genotyped by seminested multiplex PCR. Amplicons were sequenced, aligned by ClustalW, and phylogenetic analyses were conducted in MEGA6. Sequences data were deposited to GenBank and DDBJ.Medical examination and microbiological analyses upheld viral diarrhea. EIA revealed RVA and enterovirus. PCR identified virulent RVA strain GIP[6] whose VP7 nucleotide sequences shared a common cluster with Cuban isolate G1P[6], while the VP4 P[6] sequences were related to Asian strains. Reassortant RVA G1P[6] was found in fatal diarrhea cases and mortality of a Nigerian child. RVA coinfection with enterovirus and associated biomarkers of environmental enteric dysfunction in infantile diarrhea should henceforth be evaluated. Current rotavirus vaccines may fare badly against the prevailing virulent strains. The disease severity and outcome necessitates a wider epidemiological study, a review and inclusion of the P[6] genotype in future rotavirus vaccines.

Fast, Sensitive and Specific Detection of Thailand orthohantavirus and its Variants Using One-Step Real-Time Reverse-Transcription Polymerase Chain Reaction Assay.

Genetic variants of Thailand orthohantavirus (THAIV) have been recently reported from rodents in South-East Asia and in islands from the South-West part of the Indian Ocean. In order to detect THAIV and its variants, we developed a sensitive and specific real-time RT-PCR targeting the S segment. Our assay was developed in two different RT-PCR systems that gave similar results in terms of sensitivity. Moreover, our results demonstrated a specificity of 100%.