多重实时荧光定量PCR检测急性肝炎患者HBV和HCV方法的建立与意义分析

目的建立一种新的可以快速筛查急性肝炎患者HBV和HCV感染的多重实时荧光定量PCR方法。方法收集596例急性肝炎患者血清或血浆样本,应用自主构建的多重实时荧光定量PCR方法同时检测HBV和HCV,同时应用HBV和HCV单管检测试剂进行检测分析。结果新建立的多重实时荧光定量PCR体系,可以有效地检测HBV和HCV的阳性质控品及阳性样本。多重实时荧光定量PCR体系特异性较好,检测HBV和HCV时,两者之间无交叉反应,且以多种血浆病毒的核酸样品为模板进行检测时结果全为阴性;多重实时荧光定量PCR体系检测HBV、HCV的灵敏度分别为20 IU/ml、50 IU/ml;多重实时荧光定量PCR体系精密度好,检测HBV、HCV的CV值分别为0.11%、0.096%。在检测临床样本时,多重复合体系检测血清或血浆中HBV和HCV的检出率与单独检测HBV和HCV具有很好的一致性,且HBV和HCV的一致率分别达到97.7%(k=0.952)、98.8%(k=0.976);同时有6例样本出现HBV和HCV共感染。结论新型多重实时荧光定量PCR方法对快速检测及筛查HBV和HCV较快捷,而且特异性好、灵敏度高、精密度好。因此多重实时荧光定量PCR方法对2种肝炎病毒的检测具有重要意义。

A novel multiplex real-time PCR assay established for the concurrent detection of hepatitis B and C viruses in patients with acute hepatitis

Objective A novel multiplex real-time PCR assay for concurrent detection of hepatitis viruses was evaluated for its clinical performance in screening patients with acute hepatitis. Methods A total of 596 serum or plasma samples were collected from patients with acute symptoms of hepatitis. Concurrent detection of nucleic acids of HBV and HCV was performed using the multiplex real-time PCR. At the same time,single real-time PCR was also used to do the detection test. Results The novel multiplex real-time PCR assay could effectively detected HBV and HCV with good specificity, showing no cross reactions with each other and no false positive results for negative control samples. The sensitivities for detecting HBV and HCV were 20 IU/ml, and 50 IU/ml, respectively.The accuracy of the method was good, with the CV values for detecting HBV and HCV were 0.11%, and 0.096% ,respectively. Concordance rates between the results from the multiplex real-time PCR and the single real-time PCR tests for the detection of HBV and HCV were 97.7% （k=0.952）,and 98.8%（k=0.976）. The method was used to screen the clinical samples. 6（1.0%） were positive for both HBV and HCV. Conclusion The novel multiplex real-time PCR test is good, not only fast, but also convenient,with good specificity,high sensitivity,and good precisian.Therefore,this new multiplex PCR assay has great importance for the concurrent detection of the two hepatitis viruses.