Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

Abstract

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

“…RPA, another isothermal amplification assay, requires only two primers, is carried out at a lower temperature (39°C) and takes shorter time (<30 min). Due to its advantages compared to other isothermal amplification methods, the RPA method has been widely used for rapid detection of multiple pathogens that infected with human beings and animals (Chen et al., ; Ma, Zeng, Huang, et al., ; Moore & Jaykus, ; Vasileva Wand, Bonney, Watson, Graham, & Hewson, ; Wang, Zhao, et al., ). For point‐of‐care diagnostics in remote area, RPA method is of great interest due to its portability, short turnaround time and potential for construction of a mobile laboratory.…”

“…The sequence of N gene was highly conserved within the whole PDCoV genome, thus N gene was chose for preparing the RNA standard in this study. The in vitro transcribed RNA standard was prepared following a previously reported method (Ma, Zeng, Huang, et al., ). Nucleic acids were extracted from the cells infected with PDCoV strain CHN‐GD16‐03 (accession no: ) using a TIANamp virus DNA/RNA Kit (Tiangen Biotech, China), and used as the template in the RT‐PCR assay with a one‐step RT‐PCR Kit (Takara, China).…”

“…The loop‐mediated isothermal amplification (LAMP) assay, which required shorter run time compared with PCR/qPCR, was reported for the detection of PDCoV (Hanaki et al., ). In comparison to LAMP assay, the recombinase polymerase amplification (RPA) assay, which required fewer primers (two primers) and shorter run time (20–30 min), has been widely reported in pathogen detection (Lillis et al., ; Ma, Zeng, Huang, et al., ; Ma, Cong, et al., ; Yang et al., ; Yang, Qin, Sun, et al., ). The isothermal amplification of the RPA assay depends on three enzymes: a recombinase polymerase, a single‐stranded binding protein and a DNA polymerase (Piepenburg, Williams, Stemple, & Armes, ).…”

“…The isothermal amplification of the RPA assay depends on three enzymes: a recombinase polymerase, a single‐stranded binding protein and a DNA polymerase (Piepenburg, Williams, Stemple, & Armes, ). RPA amplicons can be detected by gel electrophoresis or directly visualized by lateral flow dipstick (LFD) (Yang, Qin, Song, et al., ); real‐time detection of the amplification can be achieved by adding a probe to the reaction (Ma, Zeng, Huang, et al., ).…”

Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses

“…All primers for conventional RT-PCR in this study referred to former reports [33][34][35][36][37][38][39][40][41][42].…”