<i>Taq</i>Man qRT-PCR Assay for Detecting Bovine Kobuvirus

ZHAO Runtao; Temuerbagen; GUO Yu; WU Yanan; WANG Xufen; HOU Lin; ZHANG He; ZHAO Yang; ZHANG Zhidan; ZHOU Weiguang

doi:10.19303/j.issn.1008-0384.2023.07.011

Volume 38 Issue 7

Jul. 2023

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Article Navigation > Fujian Journal of Agricultural Sciences > 2023 > 38(7): 851-856

ZHAO R T, Temuerbagen, GUO Y, et al. TaqMan qRT-PCR Assay for Detecting Bovine Kobuvirus [J]. Fujian Journal of Agricultural Sciences，2023，38（7）：851−856 doi: 10.19303/j.issn.1008-0384.2023.07.011

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ZHAO R T, Temuerbagen, GUO Y, et al. TaqMan qRT-PCR Assay for Detecting Bovine Kobuvirus [J]. Fujian Journal of Agricultural Sciences，2023，38（7）：851−856 doi: 10.19303/j.issn.1008-0384.2023.07.011

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TaqMan qRT-PCR Assay for Detecting Bovine Kobuvirus

doi: 10.19303/j.issn.1008-0384.2023.07.011

1.
College of Veterinary Medicine, Inner Mongolia Agricultural University/Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases, Ministry of Agriculture and Rural Affairs, Hohhot, Inner Mongolia　010018, China
2.
Inner Mongolia Animal Disease Control Center, Hohhot, Inner Mongolia　010020, China

Received Date: 2022-10-31
Rev Recd Date: 2023-04-10

Available Online: 2023-08-16

Publish Date: 2023-07-28

Abstract

Abstract

Objective A rapid, accurate qRT-PCR method for detecting bovine kobuvirus (BKoV) was established and clinically tested for application. Method A pair of specific primers and a probe were designed and synthesized according to the 3D gene sequence of BKoV published on GenBank for the establishment of a TaqMan fluorescence qRT-PCR assay. The reaction system of the analytic was optimized prior to verification on clinical specimens. Result The optimal upstream and downstream primers were 300 nmol·L⁻¹ in concentration, and the applied probe 400 nmol·L⁻¹. A linearity with a correlation coefficient of R²= 0.999 was achieved in the range of 1×10¹−1×10⁸ copies·μL⁻¹. The amplification efficiency reached 103%. The assay demonstrated a high specificity among the pathogens related to diarrhea in calves, a high sensitivity with the minimum detection limit of 1×10¹ copies·μL⁻¹ on BKoV plasmid standard, in comparison to that of 1×10² copies·μL⁻¹ by conventional RT-PCR, and a high repeatability with a coefficient of variation of less than 3% within and between groups. In 37 bovine fecal samples collected from different pastures in Inner Mongolia from March to May 2021, the assay showed a positive detection rate of 24.3%. The calculated viral loads using the standard curve had an average on the feces from diarrheal animals at 3.7×10⁵ copies·μL⁻¹ and at 8.65×10³ copies·μL⁻¹ on the specimens from healthy calves. Conclusion The newly developed TaqMan fluorescent qRT-PCR assay was specific, repeatable, and sensitive in detecting BKoV. It could be applied for clinic diagnosis and epidemiological investigation on the disease.
- Bovine kobuvirus,
- fluorescence qRT-PCR,
- calf diarrhea,
- specificity,
- sensitivity,
- repeatability

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References(16)

References

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