实时荧光定量RT-PCR法定量检测手足口病病原体的探讨

目的了解北京市门头沟区手足口病患儿感染病毒的型别,探索用荧光定量RT—PCR法对手足口病患者咽拭子标本中肠道病毒71型（EV71）、柯萨奇病毒A16（CoxA16）型病毒载量进行定量检测的可行性。方法采用实时荧光RT—PCR体外扩增法对81例手足口病患儿咽拭子标本提取的RNA进行检测。结果28例手足口病患者体内CoxA16病毒载量〉10^3 copies·ml-1。实时荧光定量RT—PCR法构建的标准曲线显示,样本阈值环数（Ct）值与病毒拷贝数的对数（10g10）之间的相关系数为0．9998,相关性良好。4例手足口病患者咽拭子标本中EV71型病毒载量〉10^3 copies·ml-1。实时荧光定量RT-PCR法标准曲线显示,Ct值与病毒拷贝数的对数之间的相关系数为0．9996,相关性较好。结论门头沟区手足口病病原谱以CoxA16型为主,34．6％的手足口病患儿CoxA16型病毒载量〉10^3 copies·ml-1,4．9％的患儿EV71型〉10^3 copies·ml-1。实时荧光定量RT-PCR法对咽拭子标本中CoxA16、EV71核酸定量检测比较简便快速、结果直观、稳定性强,为进一步探讨患者体内病毒载量与临床症状的关系打下了良好的基础。     

16S rRNA基因检测在儿童化脓性脑膜炎早期诊断中的研究

目的探讨儿童化脓性脑膜炎快速诊断方法。方法收集可疑化脓性脑膜炎患儿脑脊液标本85例,分别用16S rRNA基因PCR方法和细菌培养法进行检测,比较两种方法检测病原菌的敏感性。结果85例标本中,PCR方法检测出33例,阳性率为38.82%,培养法检出14例,阳性率为16.47%,PCR方法检出率明显高于培养法（P〈0.01）。结论PCR方法能特异、敏感、快速地检测脑脊液感染的常见病原菌。     

Taqman荧光定量RT—PCR在呼吸道感染患者临床标本非SARS冠状病毒检测中的应用

目的建立检测四种常见人非SARS冠状病毒核酸特异的快速、敏感的TaqManqRT—PCR检测方法,应用于急性呼吸道感染患儿的感染分析。方法分别应用TaqManqRT—PCR与普通RT—PCR平行检测248份呼吸道标本,对方法的灵敏性、特异性和稳定性以及临床标本的适用性进行比较评价,阳性标本以体外转录RNA为标准品进行病毒载量定量。结果本方法可对HKU1、NL63、229E、OC43四种冠状病毒进行特异性诊断,与其他病毒无交叉反应,检测灵敏度可达10拷贝／μl,检测线性范围可达10^1～10^8拷贝／μl,248份标本中HKU1、NL63、229E、OC43阳性率依次为1．2％,0．8％,1．2％,1．6％,其中OC43荧光RT—PCR法检出率高于普通RT—PCR,其余三种病毒两种方法检测结果一致。非SARS冠状病毒阳性标本均检出于12月至次年5月。结论建立的TaqManRealtimeRT-PCR法具有特异性强、灵敏性高的特点,是开展非SARS冠状病毒的临床检测与疾病监测的有效技术手段。     

聚合酶链反应检测新生儿尿巨细胞病毒DNA

采用聚合酶链反应技术（PCR），对45例疑为巨细胞病毒（HCMV）感染的新生儿，测定其尿中的HCMV－DNA，同时与尿培养法进行比较．结果前者阳性为44．44％，后者为17．78％（P＜0．01）。从而证实PCR法具敏感、特异、简便、可靠等优点，达到对HCMV感染的早期诊断和及早干预的目的。     

病毒性脑炎

病毒、细菌、原虫、寄生虫等多种病原体均可引起脑组织炎症。病毒性脑炎（Virus encephalitis）是指病毒感染所引起的脑实质炎症，常表现为发热、头痛、抽搐、意识障碍和脑膜刺激症状等，可致中枢神经系统局灶性损害。病毒性脑炎预后不佳，死亡率高，常留有严重后遗症，如乙型脑炎患者的后遗症可达30％。病毒也可感染脑膜出现脑膜炎（Meningitis）。脑膜炎可分为细菌性和无菌性两类。后者是指脑脊液涂片和细菌培养为隐性的脑膜炎。一般无菌性脑膜炎多为病毒感染所致，故无菌性脑膜炎和病毒性脑膜炎几乎成为同意词。病毒性脑膜炎的病程一般较短，预后较好。病毒性脑膜脑炎（Meningoencephalitis）是指脑实质和脑膜同时感染。     

半套式逆转录聚合酶链反应技术诊断中枢神经系统肠道病毒感染

肠道病毒 (ＥＶ)感染常见 ,所致疾病轻重不一 ,可表现为无症状感染或严重的脑膜炎、脑炎和心肌炎等。长期以来缺乏有效的病原学诊断方法。组织培养分离病毒费时 ,阳性率低 ,且病合 1至 2周得到结果时已无益于临床诊断和治疗。聚合酶链反应 (ＰＣＲ)方法能快速、特异地检测出脑膜炎病人脑脊液 (ＣＳＦ)中ＥＶＲＮＡ ,但感染初期及回顾性研究的临床标本中病毒滴度较低 ,扩增结果多为阴性 ,病毒培养也常常失败。我们采用二步扩增半套式ＰＣＲ方法检测无菌性脑膜炎病人脑脊液 (ＣＳＦ)和血清标本中肠道病毒ＲＮＡ ,发现该方法较常规一步ＰＣＲ敏…     

2009年夏季174例手足口病患儿病原分析

目的分析本院2009年夏季手足口病患儿病原及其血清型特征,为临床早期诊断和疾病防控提供实验依据。方法2009年4—9月,首都儿科研究所附属儿童医院门诊就诊的174例手足口病患儿咽拭子和疱疹液,实时荧光RT—PCR方法检测肠道病毒通用型（EV）、柯萨奇A16型（CA16）、肠道病毒71型（EV71）。131例患儿双份血清,同时检测CA16、EV71IgM抗体。CA16和EV71阳性标本扩增VP1区基因片段后测序,进行同源性和系统进化分析。结果（1）EV、CA16、EV71阳性例数分别为167、112、46;阳性率为96．O％、64．4％、26．4％,CA16：EV71为2．43：1。（2）首诊血清CA16和EV71IgM阳性例数为51、25,阳性率为38．9％、19．1％,复诊阳性例数为98、32,阳性率为74．8％、24．4％。（3）CA16VP1区核苷酸同源性为88．7％-98．5％,EV71VP1区核苷酸同源性为94．9％-99．7％,与c4亚型参比序列核苷酸同源性92．1％～95．3％。结论2009年夏季本院手足口病患儿病原以CA16、EV71为主,EV71阳性率较之前报道有较大幅度升高。EV71病毒株以c4亚型为主。实时RT-PCR法较血清学检测特异性IgM抗体更适于疾病早期诊断。     

A组轮状病毒一步法RT—PCR检测技术的建立和应用研究

目的 设计A组轮状病毒（RV）的特异性引物,应用Tth酶,建立一步法RT－PCR扩增方案,检测西安地区腹泻幼儿196份粪便标本,并与本室研制建立的反向间接血凝法（RPHA）,聚丙烯酰胺凝胶电泳,市售ELISA试剂盒平行检测对比分析。方法 设计并合成第九基因序列保守区互补的特异性引物,在经典法RT－PCR试验成功的基础上,建立合理的一步法RT－PCR＞要四种检测结果阳性率分别为33．16％,23．47％,21．94％和25％,X^2检验表明,RT－PCR最为敏感。结论 反转录和PCR由Tth酶一步完成,克服了经典法中AMV、RNasin等试剂昂贵,操作繁锁等缺点,很适合临检需要。     

肾综合征出血热患者血清汉坦病毒基因的PCR-ELISA方法建立

目的：建立检测肾综合征出血热（HFRS）患者血清标本中HV基因的PCR－ELISA方法。方法：设计并合成互补于HV S基因片段的标记引物。对108例HFRS患者血清进行巢式RT－PCR扩增，并用酶免方法分析扩增产物。结果：108例患者不同病日采集的血清标本的巢式RT－PCR平均检出率仅为62．0％，而PCR－ELSA平均检出率为81．5％，结论：PCR－ELISA用于早期HFRS患者的HV基因检测是一种理想的检测方法。     

单克隆抗体ELISA法快速诊断疱疹性脑炎

本文用单纯疱疹病毒单克隆抗体（ＨＳＶ－ＭｃＡｂ）ＥＬＩＳＡ法检测了１２８例病毒性脑炎患儿脑脊液中ＨＳＶ特异性抗原（ＨＳＶ—Ａｇ），２０例其他神经系统疾患也作了测定，３２例脑炎患儿同时取脑脊液和血清作ＨＳＶ—ＩｇＧ抗体水平测定比较，病毒性脑炎患儿的ＨＳＶ－Ａｇ阳性检出率为１９．５％，与血清／脑脊液的ＨＳＶ－ＩｇＧ测定比较，敏感性８４．６％，特异性９４．７％。用ＥＬＩＳＡ法检测脑脊液中ＨＳＶ－Ａｇ是一种简便、快速、敏感、特异的方法，对疱疹性脑炎具有早期诊断价值。     

2001年徐州地区暴发性病毒性脑膜炎病原的研究

目的 确定引起2001年江苏省徐州地区无菌性脑膜炎流行的病原体。方法 组织培养法从患者脑脊液分离病毒，标准血清中和试验鉴定分离毒株；中和试验检测双份血清中和抗体效价；逆转录聚合酶链反应（RT-PCR）检测肠道病毒特异性基因片段。结果 22份脑脊液中分离出4株柯萨奇B5型、2株柯萨奇B3型、1株艾可7型肠道病毒，分离阳性率31．8％。RT-PCR检测脑脊液21份，肠道病毒阳性11份。阳性率52．4％。19例双份血清中11例中和效价呈4倍以上增长或转阳，阳性率57．9％。结论 此次江苏省徐州地区无菌性脑膜炎流行的病原体是以柯萨奇B5为主要血清型的肠道病毒。     

Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer of 2007

BackgroundAseptic meningitis is the most commonly observed CNS infection and is mainly attributed to Non-Polio Enteroviruses (EV).ObjectiveIdentification and genetic analysis of the EV involved in the recent aseptic meningitis outbreak which occurred in Greece, during the summer of 2007.Study designIn total, 213 CSF and faecal samples were examined for EV presence by culture, while enteroviral RNA detection was performed by nucleic acid sequence-based amplification assay (NASBA). EV strains were typed by seroneutralization, as well as nested RT-PCR followed by VP1-2A gene partial sequencing. Phylogenetic analysis was carried out for the identification of the genetic relatedness among the isolated EV strains.ResultsEV detection rate in CSF and faecal samples was 43.9% and 70.8%, respectively. EV serotyping and VP1 region analysis revealed the predominance of echovirus 4 (ECV4) serotype and the circulation of ECV6, 9, 14, 25, Coxsackie A6, A15, A24 and Coxsackie B1 serotypes. All ECV4 isolates presented a 98.7% similarity in nucleotide sequence, with a Spanish ECV4 strain, isolated during a meningitis outbreak in 2006.ConclusionsIt is the first time that ECV4 is associated with an aseptic meningitis outbreak in Greece, during which 9 different EV serotypes were co-circulating. All Greek ECV4 isolates were closely related to the Spanish ECV4 strain. Genetic analysis of the VP1 gene can significantly contribute to the revelation of the endemic EV strains circulation pattern and their phylogenetic relationship with enteroviruses involved in epidemics of distant geographical areas at different time periods.     

Detection of enterovirus RNA in cerebrospinal fluid (CSF) using NucliSens EasyQ Enterovirus assay.

Rapid detection of enterovirus (EV) infections is essential in the management of aseptic meningitis. Molecular approaches have opened the way to such rapid, but also specific and sensitive, diagnostic tests. The aim of this study was to compare the performance of the CE marked NucliSens EasyQ Enterovirus assay with an in-house two-step RT-PCR assay using cerebrospinal fluid (CSF) and throat swab samples. In addition, specificity was tested with clinical isolates positive for viruses with clinical importance in CSF samples. For nucleic acid extraction, the NucliSens miniMAG and NucliSens magnetic extraction reagents were used. Subsequently real-time nucleic acid sequence-based amplification (NASBA) RNA amplification was performed using NucliSens EasyQ basic kit reagents and NucliSens EasyQ Enterovirus reagents. An EV-specific internal homologous control (IC) RNA was used to monitor the entire NucliSens EasyQ procedure at the individual sample level. No IC but an external inhibition control was available for the RT-PCR method. For the NucliSens EasyQ procedure, amplification and real-time detection reactions were carried out in the NucliSens EasyQ analyzer. The real-time NASBA enterovirus detection was based on NASBA amplification and real-time molecular beacon technology. Data were analyzed using the manufacturer's software on the NucliSens EasyQ analyzer. For the in-house assay, RT-PCR amplicons were detected using agarose gel analysis. The analysis of clinical samples positive for HSV-1, HSV-2, adenovirus, CMV, VZV, mumps and rhinovirus were all negative by NucliSens EasyQ Enterovirus assay. Three rhinovirus samples were, however, strongly positive in RT-PCR. A total of 141 clinical samples were retrospectively tested, including 126 cerebrospinal fluid (CSF) samples and 15 throat swabs. The 91 CSF samples were negative by both methods, 31 CSF samples and 14 throat swab samples were positive by both methods. The four CSF samples were positive by RT-PCR only. One throat swab sample was negative in NucliSens EasyQ but positive in RT-PCR. The sensitivity and specificity of both methods seem to be more or less comparable. However, the in-house RT-PCR assay appears to amplify some rhinovirus strains and should therefore not be used for throat swab samples. NucliSens EasyQ Enterovirus assay gave more invalid results than the in-house RT-PCR, which is obvious taken into account the difference in quality control between the CE marked NucliSens EasyQ Enterovirus assay and the in-house enterovirus assay. The NucliSens EasyQ procedure can be completed within 5h versus 9.5h for the RT-PCR. NucliSens EasyQ Enterovirus assay showed to be a standardized, rapid, specific, sensitive and reliable procedure for the detection of enterovirus RNA.     

Clinical validation of a new real-time PCR assay for detection of enteroviruses and parechoviruses, and implications for diagnostic procedures.

BACKGROUND: Enteroviruses (EV) and parechoviruses (HPeV) are the most common causes of aseptic meningitis, encephalitis and sepsis-like syndrome in neonates. Detection by nucleic acid amplification methods improves patient management. OBJECTIVE: Development of a real-time PCR assay on a LightCycler for simultaneous detection of EV, HPeV and an internal control to monitor inhibition. STUDY DESIGN: We investigated the value of the new assay, prospectively, in a variety of samples from patients suspected of having viral meningitis or sepsis-like syndrome. RESULTS: The assay detected 64 EV serotypes and HPeV types 1-4. Of 186 patients, 63 (33.9%) were EV positive and 18 (9.7%) HPeV positive in one or more samples. In 43 of 159 feces and 6 of 57 throat samples viral culture and PCR were positive. With real-time PCR 27 extra EV and 19 HPeV positives were found. Blood and CSF were present from 33 patients. In 19 patients blood and CSF were positive, one was only positive in CSF, two were only positive in blood, 11 were negative. From 96 patients CSF and/or blood samples were tested and compared to results in throat and/or feces samples. Forty patients were EV-PCR and 14 HPeV-PCR positive in blood and/or CSF. All of these were confirmed by a positive PCR for the respective virus in feces and/or throat. CONCLUSIONS: Simultaneous detection of EV and HPeV with this two-step real-time PCR is specific, faster and more sensitive than viral culture. All systemic infections (blood or CSF positive) were confirmed in feces. Culture is no longer necessary for clinical diagnosis and should only be performed on PCR-positive samples to obtain isolates for typing purposes. Application of this assay is an important improvement for patient management since the outcome of the analysis is available within the time frame of clinical decision-making.     

Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.

BACKGROUND: Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. OBJECTIVE: To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. STUDY DESIGN: After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. RESULTS: Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80). CONCLUSIONS: The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.