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Article

An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control

by
Hye-Ryung Kim
1,
Jong-Min Kim
1,
Ji-Su Baek
1,
Jonghyun Park
1,
Won-Il Kim
2,
Bok Kyung Ku
3,
Hye-Young Jeoung
3,
Kyoung-Ki Lee
3 and
Choi-Kyu Park
1,*
1
College of Veterinary Medicine & Institute for Veterinary Biomedical Science, Kyungpook National University, Daegu 41566, Republic of Korea
2
College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, Republic of Korea
3
Animal and Plant Quarantine Agency, Gyeongsangbuk-do, Gimcheon 39660, Republic of Korea
*
Author to whom correspondence should be addressed.
Viruses 2023, 15(11), 2204; https://doi.org/10.3390/v15112204
Submission received: 12 October 2023 / Revised: 30 October 2023 / Accepted: 31 October 2023 / Published: 1 November 2023
(This article belongs to the Special Issue Porcine Epidemic Diarrhea Virus (PEDV): Pathogenesis and Prevention)
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Abstract

For rapid and reliable detection of porcine epidemic diarrhea virus (PEDV) from pig clinical samples, a multiplex, real-time, reverse transcription loop-mediated isothermal amplification (mqRT-LAMP) was developed using two sets of primers and assimilating probes specific to the PEDV N gene and the Sus scrofa β-actin gene, which was used as an endogenous internal positive control (EIPC) to avoid false-negative results. The assay specifically amplified both target genes of PEDV and EIPC in a single reaction without any interference but did not amplify other porcine viral nucleic acids. The limit of detection was 10 copies/μL, 100-fold lower than that of a reverse transcription-polymerase chain reaction (RT-PCR) and equivalent to that of quantitative/real-time RT-PCR (qRT-PCR). This assay has high repeatability and reproducibility with coefficients of variation < 4.0%. The positive signal of the mqRT-LAMP assay was generated within 25 min, demonstrating advantages in rapid detection of PEDV over RT-PCR or qRT-PCR assay, which require at least 2 h turnaround times. In clinical evaluation, the detection rate of PEDV by mqRT-LAMP assay (77.3%) was higher than that of RT-PCR assay (69.7%), and comparable to qRT-PCR (76.8%) with almost 100% concordance (kappa value 0.98). The developed mqRT-LAMP assay can serve as an advanced alternative method for PEDV diagnosis because it has high sensitivity and specificity, rapidity, and reliability even in resource-limited laboratories.
Keywords: PEDV; multiplex real-time RT-LAMP; assimilating probe; N gene; Sus scrofa β-actin gene; internal positive control PEDV; multiplex real-time RT-LAMP; assimilating probe; N gene; Sus scrofa β-actin gene; internal positive control

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MDPI and ACS Style

Kim, H.-R.; Kim, J.-M.; Baek, J.-S.; Park, J.; Kim, W.-I.; Ku, B.K.; Jeoung, H.-Y.; Lee, K.-K.; Park, C.-K. An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control. Viruses 2023, 15, 2204. https://doi.org/10.3390/v15112204

AMA Style

Kim H-R, Kim J-M, Baek J-S, Park J, Kim W-I, Ku BK, Jeoung H-Y, Lee K-K, Park C-K. An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control. Viruses. 2023; 15(11):2204. https://doi.org/10.3390/v15112204

Chicago/Turabian Style

Kim, Hye-Ryung, Jong-Min Kim, Ji-Su Baek, Jonghyun Park, Won-Il Kim, Bok Kyung Ku, Hye-Young Jeoung, Kyoung-Ki Lee, and Choi-Kyu Park. 2023. "An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control" Viruses 15, no. 11: 2204. https://doi.org/10.3390/v15112204

APA Style

Kim, H.-R., Kim, J.-M., Baek, J.-S., Park, J., Kim, W.-I., Ku, B. K., Jeoung, H.-Y., Lee, K.-K., & Park, C.-K. (2023). An Advanced Multiplex Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Porcine Epidemic Diarrhea Virus and Porcine Internal Positive Control. Viruses, 15(11), 2204. https://doi.org/10.3390/v15112204

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