A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection and Nucleic Acid Extraction
2.2. Primer and Probe Design of RT-RAP Assay
2.3. Plasmid Construction
2.4. Solid Docosane Barrier
2.5. One-Pot Two-Stage RT-RAP Assay
2.6. Sensitivity, Specificity, and Reproducibility of the RT-RAP Assay
2.7. RT-RAP Detection of Clinical Samples
2.8. Statistical Analysis
3. Results
3.1. Optimization of the One-Pot Two-Stage RT-RAP Assays
3.2. Sensitivity, Specificity, and Reproducibility of the RT-RAP Assay
3.3. Viral Loads of Clinical Samples
3.4. Clinical Evaluation and Comparison between the RT-RAP and a Commercial qRT-PCR Kit
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Region | Primer/Probe | Sequence (5′—3′) | Source | Position a |
---|---|---|---|---|
Gag | HIV-RAA-F | GAAGTAATACCCATGTTTTCAGCATTATCA | This paper | 1288–1317 |
HIV-RAA-R | TGCAGCTTCCTCATTGATGGTTTCTTTTAAC | This paper | 1419–1389 | |
HIV-qPCR-F | CATGTTTTCAGCATTATCAGAAGG | [9] | 1299–1322 | |
HIV-qPCR-R | GCTTCCTCATTGATGGTCTCTTT | [9] | 1415–1393 | |
HIV-qPCR-P | FAM-CACCCCACAAGATTTAAACACCATGCTAA-BHQ1 | [9] | 1326–1354 | |
Pol | HIV-RAA-F | ATTTTCGGGTTTATTACAGGGACAGCAGAGA | This paper | 4894–4924 |
HIV-RAA-R | CACAATCATCACCTGCCATCTGTTTTCCAT | This paper | 5070–5041 | |
HIV-qPCR-F | GGTTTATTACAGGGACAGCAGAGA | [10] | 4901–4924 | |
HIV-qPCR-R | ACCTGCCATCTGTTTTCCATA | [10] | 5060–5040 | |
HIV-qPCR-P | FAM-ACTACTGCCCCTTCACCTTTCCAGAG-BHQ1 | [10] | 4978–4953 |
Copies/Reaction | No. of Positive Samples Tested by the RT-RAP Assays for HIV-1 | ||||
---|---|---|---|---|---|
CRF_01AE | CRF_07BC | Subtype B | Subtype C/BC | CRF_08BC | |
1 | 1/8 | 1/8 | 1/8 | 2/8 | 1/8 |
101 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
102 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
103 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
104 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
105 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
Detection limit (Probit analysis) | 5.037 | 5.037 | 5.037 | 4.149 | 5.037 |
Copies/Reaction | No. of Positive Samples | Ct Values of the Commercial qRT-PCR Kits |
---|---|---|
10 | 7/8 | 36.530 |
20 | 8/8 | 35.615 |
30 | 8/8 | 33.650 |
40 | 8/8 | 34.744 |
50 | 8/8 | 34.022 |
100 | 8/8 | 33.794 |
Detection limit (Probit analysis) | 13.678 |
RT-RAP | Commercial qRT-PCR Kits | Total | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) | Kappa | |
---|---|---|---|---|---|---|---|---|
+ | − | |||||||
+ | 99 | 1 | 100 | |||||
− | 0 | 70 | 70 | 100.00 | 98.59 | 99.00 | 100 | 0.988 |
Total | 99 | 71 | 170 |
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Tian, F.; Jin, C.; Ji, S.; Tie, Y.; Fan, G.; Zhang, R.; Zheng, Y.; Shen, X.; Ma, X.; Feng, Z. A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay. Trop. Med. Infect. Dis. 2023, 8, 105. https://doi.org/10.3390/tropicalmed8020105
Tian F, Jin C, Ji S, Tie Y, Fan G, Zhang R, Zheng Y, Shen X, Ma X, Feng Z. A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay. Tropical Medicine and Infectious Disease. 2023; 8(2):105. https://doi.org/10.3390/tropicalmed8020105
Chicago/Turabian StyleTian, Fengyu, Cong Jin, Shangzhi Ji, Yanqing Tie, Guohao Fan, Ruiqing Zhang, Yehuan Zheng, Xinxin Shen, Xuejun Ma, and Zhishan Feng. 2023. "A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay" Tropical Medicine and Infectious Disease 8, no. 2: 105. https://doi.org/10.3390/tropicalmed8020105
APA StyleTian, F., Jin, C., Ji, S., Tie, Y., Fan, G., Zhang, R., Zheng, Y., Shen, X., Ma, X., & Feng, Z. (2023). A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay. Tropical Medicine and Infectious Disease, 8(2), 105. https://doi.org/10.3390/tropicalmed8020105