Department of Clinical Laboratory, Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan, 430061, Hubei, China.
Hubei Academy of Traditional Medicine, Wuhan, 430074, Hubei, China.
Virol J. 2021 Feb 18;18(1):38. doi: 10.1186/s12985-021-01510-6.
In recent years, fluorescent quantitative polymerase chain reaction assays for detecting viral DNA are in widespread use throughout the world. However, considering the wide distribution of new herpesvirus among the population, we constructed a method to detect HHV-6, 7, and 8 simultaneously.
The blood samples of 74 blood donors and 45 pityriasis rosea patients were collected. The recombinant plasmids containing U67, U36, and orf65 were constructed to optimize the PCR reaction system. The forward and reverse primers and probe sequences of HHV-6 were as follows: TAAATATCGATGCCGCTCTG, ACGTTCTAGCCATCTTCTTTG, CGCAAACGACAAAGCCA. The forward and reverse primers and probe sequences of HHV-7 were as follows: TTAGACATCTTACACGACAGC, CAGCTTTTCGAACTTGTCAC, TTCATCGGGTACGTCCA. The forward and reverse primers and probe sequences of HHV-8 were as follows: GCGACATATTTCCCTGATCC, CCAACTTTAAGGTGAGAGACC, CATGCGAGCCACCAG. Through the detection of housekeeping genes, DNA sequencing, and optimization of the PCR reaction system, the triple fluorescent quantitative PCR detection system was constructed. Blood samples of blood transfusion staff and pityriasis rosea patients were detected.
The correlations of HHV-6, 7, and 8 between single and multiplex PCR are 0.980, 0.987, 0.965, respectively. In 74 blood donor samples, 16.2% of HHV-6 and 55% of HHV-7 were positive (viral load > 3 log10 copies/ml) according to multiplex real-time PCR. In 45 patients suspected of pityriasis rosea (PR) infection, 40% HHV-6, 73.3% positive cases are found.
With the safety of blood transfusion being a major concern of the public, this method will show good specificity and sensitivity in blood transfusion screening.
近年来，荧光定量聚合酶链反应检测病毒 DNA 在世界范围内得到广泛应用。然而，考虑到新的疱疹病毒在人群中的广泛分布，我们构建了一种同时检测 HHV-6、7 和 8 的方法。
采集 74 名献血者和 45 名玫瑰糠疹患者的血液样本。构建含有 U67、U36 和 orf65 的重组质粒，优化 PCR 反应体系。HHV-6 的正向和反向引物及探针序列如下：TAAATATCGATGCCGCTCTG、ACGTTCTAGCCATCTTCTTTG、GCAAACGACAAAGCCA。HHV-7 的正向和反向引物及探针序列如下：TTAGACATCTTACACGACAGC、CAGCTTTTCGAACTTGTCAC、TTCATCGGGTACGTCCA。HHV-8 的正向和反向引物及探针序列如下：GCGACATATTTCCCTGATCC、CCAACTTTAAGGTGAGAGACC、CATGCGAGCCACCAG。通过管家基因检测、DNA 测序和 PCR 反应体系优化，构建了三重荧光定量 PCR 检测系统。检测了输血工作人员和玫瑰糠疹患者的血液样本。
单重和多重 PCR 检测 HHV-6、7 和 8 的相关性分别为 0.980、0.987 和 0.965。在 74 名献血者样本中，根据多重实时 PCR，16.2%的 HHV-6 和 55%的 HHV-7 呈阳性（病毒载量>3 log10 拷贝/ml）。在 45 名疑似玫瑰糠疹（PR）感染的患者中，发现 40%的 HHV-6 和 73.3%的阳性病例。
鉴于公众对输血安全性的高度关注，该方法在输血筛查中具有良好的特异性和敏感性。
