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

Abstract

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 10 2 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.

Quantitative PCR Assays for the Strain-Specific Identification and Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus X253

“…The holA gene encodes the delta subunit of DNA polymerase III. According to Wang et al [ 27 ], Cq values less than 35 are considered positive.…”

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex

“…One of the limitations of multiplexing is related to the overlapping emission and spectra that some florescent dyes have, which limits their use in multiplex scenarios. This issue has been overcome in recent years with a new generation of fluorescent dyes by several manufacturers [51][52][53][54].…”

A triplex real-time PCR method to detect African swine fever virus gene-deleted and wild type strains

“…PCR assay was performed on an CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with the following conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, annealing at different temperatures (55–60°C) for 45 s. Fluorescence was recorded at 59°C. Copy number was calculated using the formula (Copy number = [(6.02 × 10 23 ) × ([ng/μl] × 10 −9 )]/[DNA length × 660]) described previously ( 19 ). In addition, plasmids (ASFV-B646L-pUC57, ASFV-MGF_360-14L-pUC57, and ASFV-CD2v-pUC57) with a series of 10-fold dilution (10 −2 -10 −11 ) were used as the templates to validate the method.…”