Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus
Abstract
:1. Introduction
2. Results
2.1. RNA Standard Preparation
2.2. Precision and Reproducibility Analysis
Template Quantity (Copies/Reaction) | HAV Detection | HEV Detection | ||
---|---|---|---|---|
Ct Value (Range) | Average | Ct Value (Range) | Average | |
1,000,000 | 17.79–18.01 | 17.89 | 13.56–13.75 | 13.71 |
100,000 | 21.11–21.36 | 21.23 | 16.88–17.08 | 16.93 |
10,000 | 24.58–24.82 | 24.70 | 20.19–20.45 | 20.33 |
1000 | 27.88–28.26 | 28.08 | 23.51–23.92 | 23.76 |
100 | 31.22–31.60 | 31.42 | 26.78–27.32 | 27.02 |
10 | 34.61–35.11 | 34.83 | 29.77–30.56 | 30.35 |
2.3. Sensitivity and Linearity
Template Quantity (Copies/Reaction) | Ct Value (Average Ct for Triplicate Reactions) | |
---|---|---|
HAV Template | HEV Template | |
10,000,000 | 14.65 ± 0.09 | 10.28 ± 0.08 |
1,000,000 | 17.91±0.14 | 13.71 ± 0.08 |
100,000 | 21.29 ± 0.19 | 17.05 ± 0.12 |
10,000 | 24.78 ± 0.21 | 20.39 ± 0.18 |
1000 | 28.03 ± 0.31 | 23.73 ± 0.31 |
100 | 31.39 ± 0.34 | 27.12 ± 0.49 |
10 | 34.77 ± 0.52 | 30.54 ± 0.66 |
1 | NA | NA |
2.4. Specificity Analysis
2.5. Application of Multiplex Real-Time PCR to Clinical Sera Samples
3. Discussion
4. Methods
4.1. Ethics Issues
4.2. Clinical Specimens from Patients with Acute HAV Infection and HEV Infection
4.3. Viral RNA Extracted from Clinical Sera Specimens
4.4. RNA Standard Preparation
4.5. Primer and Probe Design for HAV-HEV Multiplex Real-Time PCR
Targeted Viruses | Name | Sequence of Oligonucleotide (5'–3') | Location | Amplicon Length |
---|---|---|---|---|
Hepatitis A Virus | Forward primer | GGT AGG CTA CGG GTG AAA C | 393~411 a | 116 nt |
Reverse primer | CCT CCG GCG TTG AAT GGT TT | 489~508 a | ||
Probe | FAM-ACA GCG GCG GAT ATT GGT GAG TTG TTA AGA-BHQ | 456~485 a | ||
Hepatitis E Virus | Forward primer | GGT GGT TTC TGG GGT GAC | 5261~5278 b | 70 nt |
Reverse primer | AGG GGT TGG TTG GAT GAA | 5313~5330 b | ||
Probe | Cy5-TGA TTC TCA GCC CTT CGC-BHQ | 5284~5301 b |
4.6. Multiplex Real-Time PCR Conditions
5. Conclusions
Acknowledgments
Conflicts of Interest
References
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Qiu, F.; Cao, J.; Su, Q.; Yi, Y.; Bi, S. Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus. Int. J. Mol. Sci. 2014, 15, 9780-9788. https://doi.org/10.3390/ijms15069780
Qiu F, Cao J, Su Q, Yi Y, Bi S. Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus. International Journal of Molecular Sciences. 2014; 15(6):9780-9788. https://doi.org/10.3390/ijms15069780
Chicago/Turabian StyleQiu, Feng, Jingyuan Cao, Qiudong Su, Yao Yi, and Shengli Bi. 2014. "Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus" International Journal of Molecular Sciences 15, no. 6: 9780-9788. https://doi.org/10.3390/ijms15069780