TaqMan real-time PCR assay for the quantification of HIV-1 DNA

doi:10.3877/cma.j.issn.1674-1358.2017.02.006

Abstract

Abstract:

Objective

To establish a real-time PCR assay for HIV DNA quantification.

Methods

The primers were designed according to the sequence of LTR-gag gene. A recombinant plasmid containing LTR-gag gene was constructed as a standard control. HIV DNA quantification were detected by real-time PCR assay. Reproducibility and specificity of this assay were also evaluated. HIV DNA from 12 cases with HIV-1 infection was also detected. The results were compared with Roche HIV DNA qualitative detection kit.

Results

The detection limit of the fluorescent quantitative PCR was 50 copies/μl. The correlation coefficient of the standard curves was 0.99, the slope was -1.65, and the intercept was 39.80. HIV-1 DNA from 12 cases with HIV-1 infection was successfully quantified using this assay. All the results were consistent with the results of the Roche HIV DNA qualitative detection kit, with no significant differences in the results of HIV DNA by different extraction methods (t = 0.033, P = 0.974).

Conclusion

HIV DNA detection method that is rapid, specific and repeatable was established successfully.

Key words: Real-time PCR, TaqMan probe, Human immunodeficiency virus-1

Genpeng Han, Yong Xiong, Yu Gao, Xiaoxia Yang. TaqMan real-time PCR assay for the quantification of HIV-1 DNA[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2017, 11(02): 129-133.

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Figures/Tables 5

References 19

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错配	NCBI数据库中所占比例（%）
错配	HIV-FOR引物	HIV探针	HIV-REV引物
None	4 881（90.82）	1 924（99.70）	1 810（97.52）
3G→A	249（4.63）	—	—
4A→G	245（4.55）	—	—
9A→G	—	5（0.30）	—
引物2	—	—	16（0.86）
13G→A	—	—	5 （0.28）
14C→T	—	—	25（1.34）

错配	NCBI数据库中所占比例（%）
错配	HIV-FOR引物	HIV探针	HIV-REV引物
None	4 881（90.82）	1 924（99.70）	1 810（97.52）
3G→A	249（4.63）	—	—
4A→G	245（4.55）	—	—
9A→G	—	5（0.30）	—
引物2	—	—	16（0.86）
13G→A	—	—	5 （0.28）
14C→T	—	—	25（1.34）

样本编号	方法1		方法2		血浆HIV-1 RNA （拷贝/ml）
样本编号	Ct值（±s）	拷贝数	Ct值（±s）	拷贝数	血浆HIV-1 RNA （拷贝/ml）
1	25.96±0.16	4 387	26.12±0.07	3 996	6 890
2	26.68±0.11	2 833	26.82±0.11	2 613	13 458
3	24.07±0.16	13 817	24.28±0.13	12 151	189 537
4	24.97±0.18	7 993	24.95±0.30	8 113	89 231
5	24.34±0.06	11 717	24.31±0.22	11 926	165 782
6	22.99±0.03	26 558	22.96±0.03	27 115	203 563
7	28.02±0.11	1 263	28.08±0.32	1 214	7 057
8	24.02±0.06	14 208	24.02±0.27	14 211	179 437
9	24.27±0.01	12 235	24.26±0.24	12 345	153 784
10	31.29±0.05	174	31.19±0.17	185	452
11	31.47±0.15	156	31.55±0.26	148	280
12	24.41±0.11	1 1260	24.40±0.08	11 317	156 783

样本编号	方法1		方法2		血浆HIV-1 RNA （拷贝/ml）
样本编号	Ct值（±s）	拷贝数	Ct值（±s）	拷贝数	血浆HIV-1 RNA （拷贝/ml）
1	25.96±0.16	4 387	26.12±0.07	3 996	6 890
2	26.68±0.11	2 833	26.82±0.11	2 613	13 458
3	24.07±0.16	13 817	24.28±0.13	12 151	189 537
4	24.97±0.18	7 993	24.95±0.30	8 113	89 231
5	24.34±0.06	11 717	24.31±0.22	11 926	165 782
6	22.99±0.03	26 558	22.96±0.03	27 115	203 563
7	28.02±0.11	1 263	28.08±0.32	1 214	7 057
8	24.02±0.06	14 208	24.02±0.27	14 211	179 437
9	24.27±0.01	12 235	24.26±0.24	12 345	153 784
10	31.29±0.05	174	31.19±0.17	185	452
11	31.47±0.15	156	31.55±0.26	148	280
12	24.41±0.11	1 1260	24.40±0.08	11 317	156 783

指标	10⁸	10⁷	10⁶	10⁵	10⁴	10³	10²
Ct值	9.86	12.46	17.35	20.46	24.56	27.91	32.71
	9.75	12.32	17.68	20.08	24.31	27.02	32.29
	9.63	12.23	17.34	20.73	24.87	27.23	32.12
Ct平均值	9.75	12.34	17.46	20.42	24.58	27.39	32.37
标准差	0.11	0.11	0.19	0.33	0.28	0.46	0.30
变异系数（%）	1.18	0.91	1.09	1.59	1.14	1.69	0.93

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