key: cord-354683-l07w3lc7
authors: Wang, Ruyi; Zhang, Wenyan; Ye, Rui; Pan, Zhongzhou; Li, Gairu; Su, Shuo
title: One-step multiplex TaqMan probe-based method for real-time PCR detection of four canine diarrhea viruses
date: 2020-06-10
journal: Mol Cell Probes
DOI: 10.1016/j.mcp.2020.101618
sha: 
doc_id: 354683
cord_uid: l07w3lc7

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 2), 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.

Viral canine diarrhea is usually highly contagious, and even more likely to cause 31 mixed infections, leading to fatal diarrhea (Williams, 1980) .. The long treatment cycle Until now, it is common laboratory practice the use of conventional polymerase chain 56 reaction (PCR) methods for single or multiplex detection. RT-PCR is a powerful 57 genetic analysis method. However, its limitations are low efficiency and the 58 requirement of sufficient concentration of virus (Ferre, 1992; Raeymaekers, 1995 Construction of standard plasmids 111 The standard fragments of the target viruses were amplified via RT-PCR and cloned into 112 the pMD18-T vector (Takara, Japan). The primer used here was identical to multiplex 113 real-time PCR method. The identity of the standard plasmids was determined by 114 sequencing of TA linkage reaction. The nucleic acid content was quantified using a Nano 115

Drop (Thermo Scientific, USA) and the copy number was calculated using the following 116 formula (1): 117

The plasmids were 10-fold serially diluted from 10 7 copies/µL to 10 1 copies/µL, and 119 standard curves and equations were prepared to verify the reliability of the dilution 120 product.

Real time PCR procedure was carried out on a LightCycler96 machine (Roche, The standard plasmid constructed according to the material method were diluted from 149 10 3 to 10 1 copies/µL to determine the limit of detection. Each concentration tested 150 three times as well as negative control. The average and standard deviation (SD) were 151 calculated. It is considered that the minimum copy concentration at which a Cq value 152 is the limit copy concentration that can be detected.

To evaluate the stability of the experiment, four different concentrations of standards 155 including 10 7 , 10 6 , 10 4 , and 10 3 copies/µL were repeated in triplicate on two separate 156 occasions with a week apart in both intra-and inter-assay as well as negative control.

The final coefficient of variation (CV) was calculated. The more stable the data, the 158 smaller the CV value. The RT-PCR was incubated in a PCR machine (Eppendorf, Germany) with a 20µ L To identify the detection limits, reactions were prepared containing 10 3 to 10 1 202 copies/µL in triplicate. Since we defined Cq values ≥ 35 as the critical point of 203 negative, the reliable detection limit was 10 2 copies/µL (Table 2) . However, the 204 average Cq value for CaKoV at 10 1 copies/µL was less than 35, there were two values 205 greater than 35 among the three replicates. Therefore, we used a conservative 206 approach and defined that the lowest detectable concentration of the virus in this 207 experiment was 10 2 copies/µL. Subsequent experiments also relies on these criteria.

The method was performed to detect canine distemper (CDV), torque teno canis virus 

Concentrations of 10 7 , 10 6 , 10 4 , and 10 3 copies/µL of standard plasmids were chosen 216 to perform three runs and measure intra-and inter-assay variation in the form of %CV.

The CV values were almost lower than 1% with a few value ranging from 1% to 218 3% (Table 3) indicating good repeatability and high accuracy. and a multiplex PCR detection developed for canine respiratory and enteric diseases.

The limit of detection of this method was also 1 × 10 4 copies/μL (Hao et al., 2019) . The data used to support the findings of this study are included within the article. 

The power of real-time PCR

Astrovirus-like, coronavirus-like, and parvovirus-like particles 435 detected in the diarrheal stools of beagle pups

Molecular epidemiological survey of canine 438 parvovirus in domestic dogs in four provinces

Isolation of Cytopathic Small Round Viruses with Bs-C-1 Cells from Patients 443 with Gastroenteritis

The authors declare no conflict of interest.

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.The limit of detection was up to 102copies/µL and performed well with high sensitivity and specificity.Our results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.