人疱疹病毒6型荧光定量分型方法的建立和临床应用

目的 建立对人疱疹病毒6型(HHV-6)能同时进行定量和分型的荧光定量PCR检测新方法,运用该方法对临床疑似病毒性脑炎患儿进行检测.方法 以HHV-6聚合酶基因区(U38)为靶序列,设计通用引物和特异性分型探针,建立能同时检测HHV-6型A/B亚型的荧光定量PCR方法,进行敏感性和特异性实验.对临床445例疑似脑炎患儿的脑脊液标本进行HHV-6荧光定量分型检测,阳性结果测序验证.结果 HHV-6A和HHV-6B病毒株荧光定鼍分型检测结果均为阳性,两亚型之间无交叉.单纯疱疹病毒1型和2型、水痘-带状疱疹病毒、巨细胞病毒、爱泼斯坦.马尔病毒、乙肝病毒、金黄色葡萄球菌、肺炎支原体、人类基因组DNA及空白对照均为阴性.HHV-6荧光定量分型最低能检测到10拷贝/μl HHV-6A/B.在临床445例疑似脑炎患儿脑脊液标本中检出HHV-6阳性21例(4.72%),其中HHV-6A阳性4例,HHV-6B阳性16例,HHV-6A和HHV-6B混合感染1例.整个PCR操作过程2-3 h.结论 HHV-6荧光定量分型方法能对HHV-6同时进行定量和分型,具有特异、敏感、简便、快速的特点,可为临床HHV-6感染性脑炎提供早期、敏感的诊断依据.

Establishment and clinical application of a new real time PCR assay for simultaneous detection of human herpesvirus-6A and human herpesvirus-6B

Objective Human herpesvirus 6(HHV-6)isolates are classified into two variants,HHV-6A and HHV-6B,based on distinct genetic,antigenic and biological characteristics.HHV-6 has been associated with encephalitis in children recently.This study aireed to estabhsh a real time PCR assay for simultaneous detection of the two subtypes of HHV-6,and apply this new assay to children with suspected encephalitis,then analyze the relationship between the infeetion with HHV-6 and encephalitis in children.Method The universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene(U38)of HHV-6.The 5'end of the probes for HHV-6A and HHV-6B was labeled with the fluoreseein reporter tetrachloro-6-carboxyfluorescein and 6-earboxyfluorescein(6-FAM),separately,while the 3'end were quenched with 6-carboxy-tetramethyl-rhedamine.The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established.Then,the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 109 to 10°copies/μl,together with controls were used for testing both sensitivity and specificity of the real time PCR assay.The cerebrospinal fluid(CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.Result Both HHV6A(strain ZJ-159)and HHV-6B (strain GS)were positive on the real time PCR assay.There were no cross-reaction with herpes simplex virus type 1,type 2(HSV-1,HSV-2),varicella-zoster virus(YZV),cytomegalovirus(CMV),EpsteinBarr virus(EBV),hepatitis B virus,Staphylococcus aureus,Mycoplasma pneumoniae and human DNA.A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6.The sensitivity threshold was 10 copies/μl for the real time PCR.HHV-6 positive rate by the real time PCR assay was 4.72%(21/445),including 4 ca8es with HHV-6A infection,16 cases of HHV-6B infeedon and l case with mixed HHV-6A and HHV-6B infeetion.The new PCR assay usually took 2 to 3 hours to provide results.Conclusion This new real time PCR assay call simultaneously detect both subtypes of HHV-6,and have high specificity and sensitivity.It will pmvide an early and sensitive diagnosis of HHV-6 encephalitis in children.