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A Multiplex PCR/LDR Assay for Viral Agents of Diarrhea with the Capacity to Genotype Rotavirus.

ABSTRACT: Rotavirus and noroviruses are major causes of diarrhea. Variable rotavirus vaccination efficacy in Africa and Asia is multifactorial, including the diversity of circulating strains and viral co-infection. We describe a multiplexed assay that detects and genotypes viruses from stool specimens. It includes a one-step reverse transcriptase PCR reaction, a ligase detection reaction (LDR), then hybridization of fluorescent products to micro-beads. In clinical samples it detects rotavirus, caliciviruses (sapovirus and norovirus), mixed infections, and genotypes or genogroups of rotaviruses and noroviruses, respectively. The assay also has the capacity to detect hepatitis A. The assay was validated on reference isolates and 296 stool specimens from the US and Ghana. The assay was 97% sensitive and 100% specific. The genogroup was concordant in 100% of norovirus, and the genotype in 91% and 89% of rotavirus G- and P-types, respectively. Two rare rotavirus strains, G6P[6] and G6P[8], were detected in stool specimens from Ghana. The high-throughput assay is sensitive, specific, and may be of utility in the epidemiological surveillance for rare and emerging viral strains post-rotavirus vaccine implementation.

SUBMITTER: Mirza AH

PROVIDER: S-EPMC6123451 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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A Multiplex PCR/LDR Assay for Viral Agents of Diarrhea with the Capacity to Genotype Rotavirus.

Mirza Aashiq H AH Das Sanchita S Pingle Maneesh R MR Rundell Mark S MS Armah George G Gyan Ben B Hodinka Richard L RL Larone Davise H DH Spitzer Eric D ED Barany Francis F Golightly Linnie M LM

Scientific reports 20180904 1

Rotavirus and noroviruses are major causes of diarrhea. Variable rotavirus vaccination efficacy in Africa and Asia is multifactorial, including the diversity of circulating strains and viral co-infection. We describe a multiplexed assay that detects and genotypes viruses from stool specimens. It includes a one-step reverse transcriptase PCR reaction, a ligase detection reaction (LDR), then hybridization of fluorescent products to micro-beads. In clinical samples it detects rotavirus, calicivirus ...[more]

PMID: 30181651

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Project description:BACKGROUND:As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS:A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS:The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS:Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.

Project description:IntroductionCalf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high.MethodsTo address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes.ResultsThis method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/μL, 96.0 copies/μL, 12.8 copies/μL, 16.4 copies/μL, 18.2 copies/μL, and 65.3 copies/μL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence.DiscussionAdditionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.

Dataset Information

A Multiplex PCR/LDR Assay for Viral Agents of Diarrhea with the Capacity to Genotype Rotavirus.

Publications

A Multiplex PCR/LDR Assay for Viral Agents of Diarrhea with the Capacity to Genotype Rotavirus.

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