One-step Multiplex PCR Research Articles

Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase PCR (RT-qPCR) to detect three nonsynonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2-positive specimens from our San Francisco Bay Area population. Between 1 December 2020 and 1 March 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K plus N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing of a validation subset of 229 specimens and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed the rapid emergence of the L452R variant in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Four species (Enterobacter cloacae, Enterobacter hormaechei, Enterobacter roggenkampii, and Enterobacter kobei) Belonging to Enterobacter cloacae Complex With Clinical Significance.

Enterobacter cloacae complex (ECC) is composed of multiple species and the taxonomic status is consecutively updated. In last decades ECC is frequently associated with multidrug resistance and become an important nosocomial pathogen. Currently, rapid and accurate identification of ECC to the species level remains a technical challenge, thus impedes our understanding of the population at the species level. Here, we aimed to develop a simple, reliable, and economical method to distinguish four epidemiologically prevalent species of ECC with clinical significance, i.e., E. cloacae, E. hormaechei, E. roggenkampii, and E. kobei. A total of 977 ECC genomes were retrieved from the GenBank, and unique gene for each species was obtained by core-genome comparisons. Four pairs of species-specific primers were designed based on the unique genes. A total of 231 ECC clinical strains were typed both by hsp60 typing and by species-specific PCRs. The specificity and sensitivity of the four species-specific PCRs ranged between 96.56% and 100% and between 76.47% and 100%, respectively. The PCR for E. cloacae showed the highest specificity and sensitivity. A one-step multiplex PCR was subsequently established by combining the species-specific primers. Additional 53 hsp60-typed ECC and 20 non-ECC isolates belonging to six species obtained from samples of patients, sewage water and feces of feeding animals were tested by the multiplex PCR. The identification results of both techniques were concordant. The multiplex PCR established in this study provides an accurate, expeditious, and cost-effective way for routine diagnosis and molecular surveillance of ECC strains at species level.

A rapid multiplex PCR assay for species identification of Asian rice planthoppers (Hemiptera: Delphacidae) and its application to early-instar nymphs in paddy fields.

Rice (Oryza sativa L.) is the main cereal crop in many Asian countries. The Asian rice planthoppers, Nilaparvata lugens (Stål) (brown planthopper), Sogatella furcifera (Horváth) (white-backed planthopper), and Laodelphax striatellus (Fallén) (small brown planthopper) (Hemiptera: Delphacidae), are the most economically important pests of rice. These three rice planthopper species often co-occur in the same paddy field. Traditionally, species identification of individuals of the three rice planthopper species has relied on morphological characters, but accurate discrimination of early-instar nymphs is very difficult, even for expert researchers. In this study, we developed a rapid one-step multiplex PCR assay using conserved and species-specific 5.8S-ITS2 rDNA gene primers for simultaneous identification of individuals of the three rice planthopper species. The multiplex PCR results showed that the three rice planthopper species could be identified accurately based on the length of the resultant amplicon, regardless of the individual developmental stage. Furthermore, we applied this assay for the first accurate quantification of early-instar nymphs of each rice planthopper species in paddy fields. Notably, we found that the species composition of early-instar nymphs cannot be extrapolated from that of adults. Thus, the multiplex PCR assay developed here facilitates detection of each rice planthopper species at the beginning of outbreaks in paddy fields.

Evaluation of one-step RT-PCR multiplex assay for body fluid identification.

The discrimination of body fluid stains provides crucial evidence during the investigation of criminal cases. Previous studies have demonstrated the practical value of mRNA profiling in body fluid identification. Conventional strategy of mRNA profiling entails reverse transcription and PCR amplification in two separate procedures with different buffer systems. In this study, we subjected the one-step multiplex reverse transcription PCR strategy to mRNA profiling with the inclusion of the same 18 tissue-specific biomarkers in the F18plex system targeting peripheral blood, menstrual blood, vaginal secretion, saliva, semen, and urine. The Qiagen OneStep RT-PCR kit and Titanium One-Step RT-PCR kit were applied to multiplex construction, while reproducible profiling results were obtained with both kits. Compared to the F18plex system, similar expression profiles of biomarkers were obtained in targeted tissues, while expected cross-reaction was observed in non-targeted body fluids. However, CYP2B7P1 and SPINK5 were detected in menstrual blood samples, which was not observed using the F18plex system. Full-profiling results were obtained in all samples using 0.1 ng peripheral blood and semen RNA, and 1 ng menstrual blood, vaginal secretion, saliva, and urine RNA. In conclusion, the application of one-step mRNA profiling strategy could be a reliable and economical method for the simplified, specific, and simultaneous analysis of tissue-specific biomarkers for the discrimination of body fluid origin.

Prevalence and genetic diversity of avian haemosporidian parasites in wild bird species of the order Columbiformes

Diseases can play a role in species decline. Among them, haemosporidian parasites, vector-transmitted protozoan parasites, are known to constitute a risk for different avian species. However, the magnitude of haemosporidian infection in wild columbiform birds, including strongly decreasing European turtle doves, is largely unknown. We examined the prevalence and diversity of haemosporidian parasites Plasmodium, Leucocytozoon and subgenera Haemoproteus and Parahaemoproteus in six species of the order Columbiformes during breeding season and migration by applying nested PCR, one-step multiplex PCR assay and microscopy. We detected infections in 109 of the 259 screened individuals (42%), including 15 distinct haemosporidian mitochondrial cytochrome b lineages, representing five H. (Haemoproteus), two H. (Parahaemoproteus), five Leucocytozoon and three Plasmodium lineages. Five of these lineages have never been described before. We discriminated between single and mixed infections and determined host species-specific prevalence for each parasite genus. Observed differences among sampled host species are discussed with reference to behavioural characteristics, including nesting and migration strategy. Our results support previous suggestions that migratory birds have a higher prevalence and diversity of blood parasites than resident or short-distance migratory species. A phylogenetic reconstruction provided evidence for H. (Haemoproteus) as well as H. (Parahaemoproteus) infections in columbiform birds. Based on microscopic examination, we quantified parasitemia, indicating the probability of negative effects on the host. This study provides a large-scale baseline description of haemosporidian infections of wild birds belonging to the order Columbiformes sampled in the northern hemisphere. The results enable the monitoring of future changes in parasite transmission areas, distribution and diversity associated with global change, posing a potential risk for declining avian species as the European turtle dove.

Fatal avian malaria in captive Atlantic puffins (Fratercula arctica) in Switzerland.

Avian malaria is a vector-borne disease caused by Plasmodium species, which may affect a broad spectrum of bird families worldwide. In most endemic and migratory birds, Plasmodium infections seem not to cause severe harm; however, non-indigenous species kept in human care such as penguins may experience high morbidity and mortality rates. Fatal avian malaria may also occur in other non-native seabirds such as puffins (Fratercula spp.), but reported cases are scarce.The aim of this study was to analyze seven cases of sudden death in captive Atlantic puffins (Fratercula arctica) at Berne Animal Park in Switzerland between 2010 and 2020, and to determine the involvement of haemosporidian parasites in the fatal outcome. In all cases, lymphoplasmacytic inflammation, necrotic lesions in several organs and presence of protozoan stages within tissues/erythrocytes or accumulation of iron-based pigment were observed histologically. A one-step multiplex PCR designed to simultaneously detect and discriminate haemosporidia belonging to the genera Plasmodium, Haemoproteus and Leucocytozoon, and a nested PCR detecting Plasmodium and Haemoproteus infections were performed on DNA extracted from formalin-fixed and paraffin-embedded (FFPE) or fresh liver and spleen tissues from five and two birds, respectively. Plasmodium spp. DNA was detected in the tissues from six of seven birds by the one-step multiplex PCR and in five of seven individuals by the nested PCR protocol. Direct sequencing of the amplification products allowed the molecular identification of Plasmodium relictum SGS1 as the involved species in three individuals and Plasmodium matutinum LINN1 in two of these fatal cases. In one bird, no haemosporidian DNA could be amplified from FFPE tissues despite of suggestive histopathological findings. These results indicate that avian malaria represents an important cause of death in captive puffins and it should be considered as a differential diagnosis in unclear or fatal cases in this threatened bird species.

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i> Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Health Indicator Bacteria That Is Useful for Risk Assessment of Peri-Implantitis

We established a novel identification method for oral Rothia species using one-step multiplex PCR analysis to investigate whether the monitoring of oral Rothia species levels is useful for peri-implantitis risk assessment, and to examine the oxygen concentration that these organisms need for growth in vitro. The mean number and proportion of Rothia aeria in peri-implant sulcus fluid (PISF) samples was significantly higher in the healthy implant group than in the peri-implantitis group (P R. aeria under aerobic conditions vigorously grew compared with those under anaerobic conditions, and this organism grew only at the upper layer where high oxygen concentrations existed in a semi-liquid nutrient medium. Therefore, the monitoring of R. aeria levels may be suitable as an indicator of healthy peri-implant tissue conditions for the prevention of peri-implantitis.

Detection of clinically important non tuberculous mycobacteria (NTM) from pulmonary samples through one-step multiplex PCR assay

BackgroundThe burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples.ResultsOut of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing.ConclusionOur study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.

One-step multiplex TaqMan probe-based method for real-time PCR detection of four canine diarrhea viruses

Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 102 copies/μL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.

Detection and differentiation of dengue virus serotypes by one-step multiplex reverse transcription PCR assays

Detection and differentiation of dengue virus serotypes by one-step multiplex reverse transcription PCR assays

First report of the stubby-root nematode Nanidorus minor infecting Paspalum vaginatum, seashore paspalum grass in Georgia, USA

We found that Nanidorus spp. was pathogenic to seashore paspalum (Paspalum vaginatum) turfgrass as its population increased from 100 to 2,080 nematodes per pot 180 days after inoculation under greenhouse conditions. Morphological measurements of adult females were similar to those described for N. minor. Molecular analysis also confirmed the morphological identification by targeting three different regions of the genomic DNA. Three primer pairs targeting 18S rDNA (360F/932R), 28S rDNA (D2A/D3B) and ITS1 rDNA (BL18/5818) were used in singleplex PCR. Forward and reverse sequences of each individual primer set were then subjected to multiple alignment and the complimentary sequences were assembled into a consensus sequence. Upon nucleotide blast on the NCBI website, they were all confirmed to be N. minor. A one-step multiplex PCR method using specific primers and a fragment size of 190 bp also confirmed the identity of N. minor. To the best of our knowledge, this is the first report of N. minor infecting seashore paspalum turfgrass in Georgia.

SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus.

This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set. The RAPD primer, OPA4, amplified a specific fragment of c. 400bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5pgµl-1 of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported CaLas-specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and CaLas simultaneously in root tissues of citrus plants. A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots. A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.

A multiplex PCR for differential detection of Echinococcus granulosus sensu stricto, Echinococcus multilocularis and Echinococcus canadensis in China

BackgroundEchinococcosis caused by Echinococcus is one of the most major infectious diseases in north-west highland of China. E. granulosus sensu strict, E. multilocularis, and E. canadensis are known to be the only three species related to human health transmitting in the areas. To achieve targeted treatment and control of echinococcosis, the accurate identification and discrimination of the species are important. However, currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency.MethodsIn the study, a set of primers were designed to aim at the three human-pathogenic Echinococcus species in China. The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity. A total of 73 parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method.ResultsThe multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products. The detection limit of the assay for each of the three species was 5 pg/μl when they were tested separately. When DNA mixtures of the targeted species containing the same concentration were used as templates, the lowest amount of DNA which can be detected was 50 pg/μl, 10 pg/μl and 5 pg/μl for E. granulosus s. s., E. multilocularis, and E. canadensis respectively. No cross-reactivity was observed when DNA from eight genetically close species was used as control templates. The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected with E. shiquicus, which showed negative signals in the developed assay. Of all the tested stool materials, 16 were previously found positive for Echinococcus by visual and microscopic examination. Among these 16 samples, 13 were confirmed by the multiplex PCR, and the other three tested negative. Additionally, the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms.ConclusionsThe developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation, which proven to be a promising tool utilized in clinic and surveillance system.

Giving More Benefits to Biosurfactants Secreted by Lactic Acid Bacteria Isolated from Plantain Wine by Using Multiplex PCR Identification

Fermented beverages have continued to give more surprises in terms of the presence of biomolecules and the diversity of microorganisms that may be contained. Republic of Congo is home to a panoply of fermented foods and beverages that are still not yet studied. This is the case of plantain wine fluently called banana wine. Within this framework, this work aims to study the role of Biosurfactant-like Biomolecules secreted during fermentation of plantain wine. Using MRS medium, 15 isolates bacteria have been found. 100% are able to secrete biosurfactant and 66.66% are extractible biosurfactants. 33% of isolates have been associated to Lactobacillus plantarum (Is2, Is9, Is12 and Is13) by using a one-step multi-plex PCR that targets genes encoding for bacteriocins. Biosurfactants secreted by L. plantarum play an important role in the preservation of banana wine. The biosurfactants extracted with chloroform and ammonium sulphate are able to inhibit the growth of pathogenic bacteria including Shigella flexneri, Salmonella spp., Pseudomonas aeruginosa and Staphy-lococcus aureus.

Developing a One-Step Multiplex PCR Assay for Rapid Detection of Four Stubby-Root Nematode Species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus.

Four trichodorid species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus, were found in multiple states in the United States. Traditional diagnosis based on morphology and morphometrics is laborious and requires an experienced taxonomist. Additionally, end-point diagnosis using PCR was only available for P. allius. To increase diagnostic efficiency and reduce costs, a one-step multiplex PCR assay was developed to simultaneously identify these four species using one PCR reaction. Available sequences of 18S ribosomal DNA and internal transcribed spacer 1 (ITS1) region of these species were aligned and five primers were designed. The conserved forward primer located in the 18S region, in combination with the species-specific antisense primer in the ITS1 region, amplified a single distinctive PCR fragment for each species (421/425 bp for P. allius, 190 bp for P. minor, 513 bp for P. porosus, and 353 bp for T. obtusus). In silico analysis with 10 other trichodorid species and experimental analysis using samples with these four species, 20 other plant-parasitic and three non-plant-parasitic nematodes demonstrated high specificity with the primers designed. The multiplex PCR amplified desirable fragments using a set of artificially mixed templates containing one, two, three, or four targeted species. The reliability of multiplex PCR results was demonstrated by using nematode populations isolated from infested fields from diverse geographic regions in eight states. The multiplex PCR-based tool developed in this study for the first time provides a simple, rapid, and cost-friendly assay for accurate diagnosis of the four major trichodorid nematodes in the United States.

Detection and Categorization of Diarrheagenic Escherichia coli with Auto-microfluidic Thin-film Chip Method

Diarrheagenic Escherichia coli (DEC) causes human diarrhea symptom in both healthy and immunocompromised individuals. An auto-microfluidic thin-film chip (AMTC) instrument integrating one-step multiplex PCR (mPCR) with reverse dot blot hybridization (RDBH) was developed for high-throughput detection of DEC. The novel mPCR method was developed by designing 14 specific primers and corresponding probes. 14 indexes including an endogenous gene (uidA) and 13 pathogenic genes (stx1, stx2, escV, ipaH, invE, estB, lt, pic, aggR, astA, bfpB, sth and stp) of DEC were detected. This one-step mPCR + RDBH approach is useful for simultaneous detection of numerous target genes in a single sample, whose specificity and availability have been confirmed on the positive control of 11 DEC strains. In addition, with 300 diarrheal stool samples being detected by this method, 21 were found to contain five major DEC strains. Compared with monoplex PCR and previous one-step mPCR approach, this method could detect ipaH and estB, and compared with current commercial kit, the relevance ratio of DEC detected by the AMTC method was increased by 1% in stool samples. Furthermore, the novel integration AMTC device could be a valuable detection tool for categorization of E. coli.

Epidemiological analysis and rapid detection by one-step multiplex PCR assay of Haemophilus influenzae in children with respiratory tract infections in Zhejiang Province, China

BackgroundHaemophilus influenzae (H. influenzae) is one of the most important pathogenic bacteria causing respiratory tract infection diseases in children. There are two main types of H. influenzae, encapsulated H. influenzae and nontypeable H. influenzae (NTHi). Serotype b of H. influenzae (Hib) used to be the main epidemic type of H. influenzae, causing the invasive infection. However, the epidemiology of invasive H. influenzae disease has changed substantially, and most invasive diseases are now caused by NTHi and other serotypes of H. influenzae. The aim of this study was to determine the main epidemic strains of H. influenzae in Zhejiang Province in China, and establish a one-step multiplex PCR assay to distinguish H. influenzae from other bacteria associated with respiratory tract infections, and distinguish encapsulated H. influenzae from NTHi.MethodIn this study, bacterial culture and serum agglutination testing were used to determine the most prevalent serotype of H. influenzae, and the results have served as a gold standard for clinical diagnosis. We also designed a one-step multiplex PCR assay using several kinds of standard strains of respiratory tract infection bacteria, to examine the stability, specificity, and detection limit of the PCR assays. We then used 1514 nasopharyngeal secretion (NPS) samples collected from children with respiratory tract infections to verify the specificity and sensitivity of the PCR assay.ResultsThe bacterial culture and serum agglutination test results showed that the positive rates of H. influenzae and encapsulated H. influenzae were 18.49 and 1.18%, respectively. The PCR results showed that the detection limit of the multiplex PCR assay was 1.89 × 103 copies /μL, the ompP6 positive rate was 19.35%, and the bexA positive rate was 1.32%. The sensitivity and specificity of the multiplex PCR were 100 and 99.86%, respectively.ConclusionsAccording to our study, the most prevalent H. influenzae subtype in Zhejiang Province was NTHi, account for 93.57%; the one-step multiplex PCR assay we established can be used as the differential detection of clinical H. influenzae strains, replacing routine bacterial culture and serum agglutination testing.

Detection of Zika, dengue and chikungunya viruses using single-reaction multiplex real-time RT-PCR

Zika (ZIKV), Dengue (DENV) and Chikungunya viruses (CHIKV) co-circulate in the same geographical areas during the same seasonal period through the same biting arthropods.Therefore a rapid sensitive and specific molecular assay for these viruses would be a considerable help in the disease management and the epidemiological survey.We developed a one-step multiplex real-time PCR for the simultaneous detection of these viruses.Intra and inter-reproducibilities varied from 0.41% to 3.29% and from 1.13% to 4.93% for each virus respectively. The specificity was 100%. Whole blood, plasma and urines were used for comparison with commercially available monoplex assays (RealStar® kits, Altona Diagnostics GmbH, Hamburg, Germany). The concordance was 96%, 92.9% and 95.7% for ZIKV, DENV and CHIKV respectively. No cross reaction and no PCR inhibition were observed for any of the clinical samples.This test can thus be used as a rapid molecular assay for ZIKV, DENV1–4 and CHIKV infections.

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR.

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.