scholarly journals One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus

2019 ◽  
Vol 31 (6) ◽  
pp. 909-912 ◽  
Author(s):  
Chunyan Jiang ◽  
Haijian He ◽  
Chaoying Zhang ◽  
Xiaoju Zhang ◽  
Jianfeng Han ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Porcine Epidemic Diarrhea Virus ◽  
Detection Method ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Reverse Transcription Pcr ◽  
Porcine Sapovirus ◽  
One Step ◽  
Porcine Epidemic Diarrhea ◽  
Porcine Sapelovirus

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 μM for PEDV, 0.15 μM for PSV, and 0.2 μM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR–based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.

scholarly journals Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

2005 ◽  
Vol 71 (4) ◽  
pp. 1870-1875 ◽  
Author(s):  
Narayanan Jothikumar ◽  
James A. Lowther ◽  
Kathleen Henshilwood ◽  
David N. Lees ◽  
Vincent R. Hill ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Nested Pcr ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Specificity And Sensitivity ◽  
Routine Monitoring ◽  
The Family ◽  
One Step ◽  
Stool Specimens ◽  
Pcr Assays

ABSTRACT Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

Direct and rapid detection of porcine epidemic diarrhea virus by RT-PCR

1997 ◽  
Vol 69 (1-2) ◽  
pp. 191-195 ◽  
Author(s):  
Kiyoyasu Ishikawa ◽  
Hideto Sekiguchi ◽  
Tomoe Ogino ◽  
Shoko Suzuki
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Rapid Detection ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea
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