Multiplex digital PCR: a superior technique to qPCR for the simultaneous detection of duck Tembusu virus, duck circovirus, and new duck reovirus

Yanwen Yin; Chenyong Xiong; Kaichuang Shi; Feng Long; Shuping Feng; Sujie Qu; Wenjun Lu; Meizhi Huang; Changhua Lin; Wenchao Sun; Zongqiang Li

doi:10.3389/fvets.2023.1222789

Multiplex digital PCR: a superior technique to qPCR for the simultaneous detection of duck Tembusu virus, duck circovirus, and new duck reovirus

Front Vet Sci. 2023 Aug 10:10:1222789. doi: 10.3389/fvets.2023.1222789. eCollection 2023.

Authors

Yanwen Yin^#¹, Chenyong Xiong^#², Kaichuang Shi¹, Feng Long¹, Shuping Feng¹, Sujie Qu¹, Wenjun Lu¹, Meizhi Huang³, Changhua Lin^{2

4}, Wenchao Sun⁵, Zongqiang Li²

Affiliations

¹ Guangxi Center for Animal Disease Control and Prevention, Nanning, China.
² College of Animal Science and Technology, Guangxi University, Nanning, China.
³ Longan Center for Animal Disease Control and Prevention, Nanning, China.
⁴ Guangxi State Farms Yongxin Animal Husbandry Group Xijiang Co., Ltd., Guigang, China.
⁵ Wenzhou Key Laboratory for Virology and Immunology, Institute of Virology, Wenzhou University, Wenzhou, China.

^# Contributed equally.

Abstract

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/μL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06-1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement.

Keywords: coefficients of variation; digital PCR; duck Tembusu virus; duck circovirus; new duck reovirus.

Grants and funding

This study was supported by the Guangxi Natural Science Foundation (Youth Science Foundation Project) (2020GXNSFBA297066), China and the Guangxi Agricultural Science and Technology Grant Project (Z201984 and Z202032), China.