增强化学发光酶免疫法测定血清促甲状腺激素方法的建及初步应用

目的 进一步提高血清促甲状腺激素（ＴＳＨ）检测的敏感性。方法 通过正交试验,并经方差分析,获得增强发光酶免疫检测法的最佳实验条件,将其与ＴＳＨ－酶联免疫吸附测定（ＥＬＩＳＡ）试剂盒相结合,建立ＴＳＨ的增强化学发光酶免疫检测法,并用此方法对５９例健康儿童和４９例甲状腺疾病患儿进行了血清ＴＳＨ的检测。结果 该方法检测人血清ＴＳＨ的灵敏度为０．０１ｍＩＵ／Ｌ。５９例健康儿童ＴＳＨ的９０％正常值范围为０．     

注意缺陷多动障碍儿童血清甲状腺激素变化的意义

目的探讨血清甲状腺激素(TH)、促甲状腺激素(TSH)在注意缺陷多动障碍(ADHD)儿童发病中的作用。方法ADHD患儿32例。其中混合型(ADHD-C)19例,注意缺陷型(ADHD-I)9例,多动-冲动型(ADHD-HI)4例。采用全自动微粒子化学发光法分别测定各组TH、TSH水平,且与15例健康儿童(对照组)进行比较。结果1.ADHD组游离三碘甲腺原氨酸(FT3)水平均正常,但与对照组相比显著升高(Pa<0.01);ADHD-I组及ADHD-HI、ADHD-C亚型组三碘甲腺原氨酸(T3)水平均正常,但与对照组相比显著升高(Pa<0.05);ADHD-I、ADHD-HI、ADHD-C组间相比,TH水平均无显著性差异(Pa>0.05)。2.ADHD组及不同亚型组TSH、游离甲状腺素(FT4)、甲状腺素(T4)水平与对照组相比,差异均无显著性意义(Pa>0.05)。结论FT3、T3可能参与ADHD的发病,TSH水平与ADHD的发病无关。     

促甲状腺激素受体基因缺陷与甲状腺疾病的研究进展

促甲状腺激素受体（TSHR）属于鸟苷酸调节偶联蛋白受体，在生理情况下，TSHR基因表达主要受促甲状腺激素调控．近年来TSHR基因缺陷所致的甲状腺功能异常已被逐步认识。TSHR基因缺陷以突变为主，引起先天性甲状腺功能亢进或毒性结节性甲状腺肿。突变分为生殖细胞突变和体细胞突变两种，多数突变的结果导致环磷酸腺苷（cAMP）信号系统激活，部分突变导致先天性甲状腺功能减低症。     

化学发光免疫分析法检测人巨细胞病毒特异性IgM抗体的研究

目的 评价LIAISON化学发光免疫分析法(CLIA)检测人巨细胞病毒(HCMV)特异性IgM抗体的方法 学特点.方法 采用CLIA法对695例婴儿肝炎综合征和病毒性肺炎患儿的血清进行HCMV-IgM检测,同时用ELISA法进行同一批样本中部分的测定.结果 共检测样本695例,其中阳性151例,阳性率21.7%.CLIA法的灵敏度可达0.82 AU/ml,批内变异系数小于8%,批间变异系数小于10%,相关性系数为0.981～0.999.两种检测方法 的一致性为92.6%,但CLIA测定敏感性显著高于ELISA法(P＜0.05).结论 CLIA法检测HCMV-IgM具有高灵敏度,检测所需时间短,人为影响因素少,结果 准确等优点,适用于临床检测.     

重视高促甲状腺素血症的诊断鉴别及处理原则

自1961年美国Guthrie医生开创新生儿筛查已有50年的历史。我国自1981年开始进行新生儿先天性甲状腺功能减低症(以下简称甲低)的筛查,目前全国筛查覆盖率超过60%[1]。大规模开展筛查以后,发现高促甲状腺素(TSH)血症的发生有所增加,部分临床医生形成了一旦发现血TSH高于正常,即加用甲状腺素替代治疗的思维模式,而对于引起高TSH血症的原因、相关检查、治疗方案及转归情况并不重视。临床误读筛查结果导致长期治疗的现象比     

单纯性热性惊厥患儿血清神经元特异性烯醇化酶、促乳素水平检测及意义

目的 探讨单纯性热性惊厥（SFC）患儿血清神经元特异性烯醇化酶（NSE）、促乳素（PRL）水平的变化，了解SFC发作后脑损伤的情况。方法 采用酶联免疫吸附法对35例单纯性热性惊厥、23例发热无惊厥（既往也无惊厥史）患儿和23例健康体检儿童进行血清NSE、PRL含量的检测。结果 SFC组血清NSE水平与发热组和健康对照组比较，差异有显著性（P〈0．05）。而发热组与健康对照组比较，差异无显著性（P〉0．05）；三组血清PRL比较，差异无显著性（P〉0．05）。结论 NSE是一个容易检测、特异性高并且稳定性强的脑损伤标志物，而PRL是判断癫痫性发作的一个有用的辅助生化指标。SFC发作时NSE有明显升高，PRL无明显变化，说明SFC发作可致脑损伤，但与癫痫发作是有区别的。     

云南省695例母亲和新生儿尿碘及新生儿血促甲状腺激素水平的调查分析

目的了解母亲尿碘与新生儿尿碘及血促甲状腺激素(TSH)水平及相关性。方法选取695例母亲及其新生儿作为研究对象,采集母亲和新生儿尿液检测尿碘,采集新生儿足跟血检测TSH。结果 695例母亲的尿碘中位数为212.9μg/L,239例(34.39%)母亲尿碘149μg/L为碘缺乏,143例(20.58%)母亲尿碘在150~249μg/L间为碘适量,163例(23.45%)母亲尿碘在250~499μg/L间为碘超足量,150例(21.58%)母亲尿碘≥500μg/L为碘过量;新生儿尿碘中位数为345.7μg/L;母亲尿碘水平与其新生儿尿碘呈正相关(r_s=0.576,P?0.001);新生儿TSH为(3.24±1.75)m IU/L,其中88例(12.66%)TSH5 m IU/L;母亲碘缺乏及碘过量的新生儿的TSH水平及TSH5 m IU/L的比例均高于母亲碘适量或碘超足量的新生儿,差异有统计学意义(P0.05)。结论本调查中母亲总体处于碘适量水平,但仍有较高比例的碘缺乏或碘过量;母亲尿碘水平与新生儿尿碘水平密切相关;碘缺乏或碘过量母亲生育高TSH新生儿的风险较高。     

内毒素对幼鼠肺等组织一氧化氮合酶免疫活性的影响

目的探讨内毒素（LPS）对幼龄大鼠肺、小肠和脾内诱导性一氧化氮合酶（iNOS）的影响。方法用免疫组织化学方法显示iNOS免疫活性表达。结果LPS可增力。肺、小肠和脾内巨噬细胞、中性粒细胞和嗜酸性粒细胞iNOS的表达。结论iNOS在不同细胞和器官的表达与注射LPS后的病理变化密切相关。     

尿促性腺激素的测定及在儿科的应用

测定尿液中促性腺激素(Gn)的含量有助于对性发育异常儿童的诊断和治疗随访。放射性免疫法是应用最早的测定尿液中Gn含量的传统方法,灵敏度通常为1IU/L。最近发展起来的以单克隆抗体为基础的非竞争性夹心固相免疫测定法和非竞争性生物素——亲和素免疫测定法的灵敏度通常为0.5IU/L,最高可达0.01 IU/L以下,能够准确检测未浓缩尿中低浓度的Gn含量。     

基于化学发光法建立深圳地区健康儿童性激素的参考区间

目的建立深圳地区0~18岁健康儿童性激素6项,即黄体生成素、促卵泡生成素、孕酮、泌乳素、雌二醇、睾酮化学发光法的参考区间值。方法采用分层整群抽样的方法于2015年9月至2016年9月在深圳市福田、罗湖、南山、宝安、龙岗5个区随机抽取0~18岁健康儿童2?178例(男童1?219例,女童959例),包括新生儿81例,婴儿335例,幼儿346例,学龄前儿童469例,学龄期儿童419例,青春期儿童528例。利用美国贝克曼DXI800化学发光仪测定黄体生成素、促卵泡生成素、孕酮、泌乳素、雌二醇、睾酮6项性激素水平。结果黄体生成素、促卵泡生成素、孕酮、泌乳素、雌二醇和睾酮在不同年龄组间水平不同,差异有统计学意义(P0.05)。同年龄组不同性别间的性激素水平的比较差异也有统计学意义(P0.05)。基于各年龄组不同性别儿童的6项激素检测水平,建立了深圳地区0~18岁健康儿童性激素6项的参考区间。结论深圳地区0~18岁健康儿童不同年龄组和不同性别组的性激素水平存在差异。该研究建立的不同性别的各年龄组的性激素6项的参考区间对儿童内分泌相关疾病的诊断与治疗有重要意义。     

人巨细胞病毒短时培养快速诊断改良技术的建立及其应用

目的 建立人巨细胞病毒（HCMV）短时培养快速诊断改良技术；检验临床样本，与病毒分离比较。方法 （1）用含细胞飞片培养板离心吸附AD169株，设时间梯度培养后用SABC法检测培养物中HCMV即刻早期抗原（IEA）；（2）用改良法检测156例患儿尿样本（7例留取脑脊液），其中54例与病毒分离对照；147例检测血清特异性IgM；（3）9例更昔洛韦（GCV）治疗后随访尿排毒和特异性IgM变化。结果 （1）标准毒株培养16－24h，能100％检出培养物中HCMV IEA。（2）快速法检测尿样本178份，阳性率46.6%。与病毒分离比较，敏感性和特异性分别达100％和91.7%。（3）147例检测血清HCMV IgM，50例阳性（34.0%），其中49例和另25例特异性IgM阴性者尿快速培养阳性，后25例中，年龄＜1岁22例；2例免疫抑制者快速培养阳性。（4）2例先天感染儿脑脊液快速培养阳性伴蛋白增高。（5）9例GCV治疗后随访2－4次，2例无效，1例尿排毒减少；6例尿排毒停止。结论 改良快速培养法敏感性高、特异性强，操作更为简便，是快速诊断活动性HCMV感染和评估抗HCMV疗效的可靠方法。     

Reference values for the concentrations of triiodothyronine, thyroxine and thyrotropin in the blood serum of euthyroid children. Method: luminescence-enhanced enzyme immunoassay

Concentrations of triiodothyronine, thyroxine and thyrotropin were determined using the luminescence enhanced enzyme immunoassay method in blood serum of 343 (male = 197, female = 146) euthyroid children. Beyond the newborn age no significant difference was found for the thyrotropin concentration. Compilation of the values resulted in a physiological thyrotropin concentration of 1.73 +/- 0.81 (1s) microU/ml for infants, children and juveniles. In opposition to these results the thyroxine and triiodothyronine concentration showed an age-specific dependence. The thyroxine and triiodothyronine concentrations (means +/- 1s) decreased continuously from the infant age (TT4: 182.1 +/- 37.7 ng/ml, 234.9 +/- 48.6 nmol/l, TT3: 1.77 +/- 0.57 ng/ml, 2.73 +/- 0.88 nmol/l) to the juvenile age (TT4: 77.9 +/- 14.3 ng/ml, 100.5 +/- 18.4 nmol/l, TT3: 1.25 +/- 0.31 ng/ml, 1.93 +/- 0.48 nmol/l). The luminescence enhanced enzyme immunoassay technique is suitable for use in the routine thyroid laboratory.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

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.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

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.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

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.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

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.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

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.     

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.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

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.