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
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples and Nucleic Acid Extraction
2.2. Construction of PEDV Reference Gene for mqRT-LAMP Assay
2.3. RT-PCR and qRT-PCR Assay
2.4. Primers and Assimilating Probes for PEDV N Gene and Internal Positive Control
2.5. Optimization of mqRT-LAMP Assay
2.6. Specificity and Sensitivity of mqRT-LAMP Assay
2.7. Precision of mqRT-LAMP
2.8. Clinical Evaluation of mqRT-LAMP
3. Results
3.1. Optimization of the mqRT-LAMP Assay
3.2. Specificity of the mqRT-LAMP Assay
3.3. Sensitivity Comparison of mqRT-LAMP with RT-PCR and qRT-PCR Assay
3.4. Precision of mqRT-LAMP
3.5. Clinical Evaluation of mqRT-LAMP
3.6. Comparative Analysis of Reaction Speed According to Clinical Evaluation Results of mqRT-LAMP and qRT-PCR
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Assay | Primer and Probe | Sequence (5′-3′) | Target Gene | Genome Position a | Reference |
---|---|---|---|---|---|
mqRT -LAMP | F3 | CTTCGAARGAACGTGACCT | PEDV N | 27,184–27,202 | [26] & this study |
B3 | CAATGCTGCAACATTTGGT | 27,356–27,374 | |||
LF | GCTATTTTCGCCCTTGGGA | 27,230–27,248 | |||
LB | AGGTGTTGATGCSTCAGG | 27,314–27,331 | |||
FIP (F1c + F2) | TGGGTCCGAAGCAAGCTG+ AGACATCCCAGAGTGGAGG | 27,253–27,270 + 27,206–27,224 | |||
BIP (B1c + B2) | TTGGAGATGCGGAATTTGTCG+ AACTGGCGATCTGAGCATAG | 27,289–27,309 + 27,332–27,351 | |||
PEDV-LF-F | FAM-ATAAGGTCCTCGCCGCTCAAGATAGGCAGA- GCTATTTTCGCCCTTGGGA | ||||
Q | TCTGCCTATCTTGAGCGGCGAGGACCTTAT-BHQ1 | ||||
EIPC-F3 | CATCCTGCGTCTGGACCT | Sus scrofa β-actin | 609–626 | This study | |
EIPC-B3 | AGCTCTTCTCCAGGGAGG | 788–805 | |||
EIPC-LF | CGCTCCGTCAGGATCTTCAT | 655–674 | |||
EIPC-LB | CTACGTCGCCCTGGACTTC | 738–756 | |||
EIPC-FIP (F1c + F2) | CCGTGGTGGTGAAGCTGTAGC+ GGACCTGACCGACTACCTC | 677–679 + 636–654 | |||
EIPC-BIP (B1c + B2) | AGATCGTGCGGGACATCAAGG+ AGTGGCCATCTCCTGCTC | 707–727 + 757–774 | |||
EIPC-LF-F | HEX-ATAAGGTCCTCGCCGCTCAAGATAGGCAGA- CGCTCCGTCAGGATCTTCAT | ||||
Q | TCTGCCTATCTTGAGCGGCGAGGACCTTAT-BHQ1 | ||||
RT- PCR | P1 | TTCCCAGCGTAGTTGAGATTG | PEDV N | 26,761–26,781 | [25] |
P2 | CGAAGTGGCTCTGGATTTGTT | 27,168–27,188 | |||
qRT- PCR | NF | CGCAAAGACTGAACCCACTAA | PEDV N | 26,684–26,704 | [13] modified |
NR | TTGCCTCTGTTGTTACTTGGAGAT | 26,858–26,881 | |||
NP-FAM | FAM–TGTTGCCATTRCCACGACTCCTGC–BHQ1 | 26,824–26,847 |
Pathogen | Strain | Source a | Amplification of Target Gene (Tp) | |
---|---|---|---|---|
PEDV N Gene (FAM) | EIPC b (HEX) | |||
Porcine epidemic diarrhea virus | KNU141112-S DEL5/ORF3 | CVAS | 9.69 | − |
Transmissible gastroenteritis virus | 175Lvac | APQA | − | 15.8 |
Porcine delta coronavirus | KNU16-07 | IVBS | − | 16.6 |
Porcine rotavirus | A1Va | IVBS | − | − |
Porcine circovirus type 2 | PCK0201 | IVBS | − | 11.2 |
Porcine reproductive and respiratory syndrome virus type 1 | Lelystad | APQA | − | − |
Porcine reproductive and respiratory syndrome virus type 2 | LMY | APQA | − | − |
Classical swine fever virus | LOM | APQA | − | 21.8 |
Swine influenza virus | VDS1 | IVBS | − | − |
Porcine parvovirus | NADL-2 | IVBS | − | 14 |
Aujeszky’s disease virus | YS | IVBS | − | 12.7 |
Non-infected pig fecal sample | − | IVBS | − | 13.7 |
Non-infected pig intestinal sample | − | IVBS | − | 11.4 |
PK-15 cell | − | IVBS | − | 10.9 |
Vero cell | − | IVBS | − | − |
Dilution (Copies/µL) | Intra-Assay Tp Value (min) | Inter-Assay Tp Value (min) | ||||
---|---|---|---|---|---|---|
High (106) | Medium (104) | Low (102) | High (106) | Medium (104) | Low (102) | |
Mean | 8.73 | 10.47 | 15.04 | 9.09 | 11.20 | 17.45 |
SD | 0.23 | 0.39 | 0.49 | 0.09 | 0.09 | 0.18 |
CV (%) | 2.65 | 3.76 | 3.28 | 0.95 | 1.74 | 1.01 |
Method | mqRT-LAMP Assay | Positive Rate | Overall Agreement (%) | |||
---|---|---|---|---|---|---|
Positive | Negative a | Total | ||||
qRT-PCR | Positive | 142 | 0 | 142 | 76.8% | 99.5% |
Negative | 1 | 42 | 43 | |||
Total | 143 | 42 | 185 | |||
RT-PCR | Positive | 129 | 0 | 129 | 69.7% | 92.4% |
Negative | 14 | 42 | 56 | |||
Total | 143 | 42 | 185 | |||
Positive rate | 77.3% |
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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
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 StyleKim, 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