单纯疱疹病毒的聚合酶链反应——微孔板反向杂交检测和分型

单纯疱疹病毒 2型 (HSV－ 2)和单纯疱疹病毒 1型 (HSV－ 1)引起的生殖器疱疹的预后不同 [1]，检测 HSV时有必要对其分型。目前，生殖器疱疹的实验室诊断“金标准”是 HSV分离培养法，该法敏感性欠佳，费时费力。新近出现的 PCR－ ELISA及 PCR－微孔板杂交法，将 PCR、核酸杂交及酶联免疫技术结合起来，克服了传统方法的诸多弊端 [2,3]。我们建立了 HSV的 PCR－微孔板反向杂交检测和分型方法，并初步评价其在生殖器标本 HSV检测和分型中的应用价值。 一、材料与方法 (一 )材料： Vero细胞自中国科学院上海细胞生物研究所引…     

细胞培养与尿液聚合酶链反应-微孔板杂交法检测沙眼衣原体感染

目的 为了建立以尿液为标本的检测沙眼衣原体的聚合酶链反应（ＰＣＲ）－微孔板杂交法。方法 以１００例性病门诊就诊者的尿液和尿道（男性）或宫颈（女性）标本，分别作沙眼衣原体ＰＣＲ－微孔板杂交法和细胞培养。两者结果不一致时用第二种不同引物作ＰＣＲ。结果 与“扩大金标准”比较，尿液ＰＣＲ－微孔板杂交法的敏感性、特异性、阳性预期值和阴性预期值分别为８０．０％、９７．３％、９０．９％和９３．６％；细胞培养的敏感性、特异性、阳性预期值和阴性预期值分别为８４．０％、１００．０％、１００．０％和９４．９％。结论 尿液ＰＣＲ－微孔板杂交法检测沙眼衣原体感染的技术性能与细胞培养相当，但有取材方便、快速的特点、将其用于性病门诊就诊者的诊断是可行的。     

生殖器疱疹患者尿道和宫颈HSV感染的研究

目的：了解生殖器疱疹（GH）患者伴发宫颈和男性尿道单纯疱疹病毒（HSV）感染情况，进一步探讨HSV和非淋菌性尿道炎／官颈炎（NGU）的关系。方法：用HSV聚合酶链反应（PCR）方法检测了56例GH患者的宫颈和男性尿道拭子标本。结果：56例患者中HSV PCR检测阳性共5例，阳性率为8．93％（5／56）。20例女性生殖器疱疹患者宫颈HSV PCR检测阳性率为25．0％（5／20）；36例男性GH患者尿道HSV PCR检测结果均为阴性。结论：生殖器疱疹患者伴发女性宫颈HSV感染更为常见，而伴发男性尿道HSV感染相对少见。HSV感染可能是非淋菌性尿道炎／宫颈炎的一个致病因子，在女性宫颈炎的发病中可能更有意义。     

多聚酶链反应与酶标法检测生殖器疱疹病毒感染的比较研究

为探讨多聚酶链反应(PCR)与酶标法检测生殖器疱疹病毒感染的诊断价值。采用PCR和酶标法检测生殖器疱疹病毒感染者45例，并用PCR作病毒分型。结果：临床确诊的45例患者中，酶标法检测抗原HSV阳性27例，阳性率60．0%(27／45)；PCR检测HSV阳性42例，阳性率93．33％(42／45)．其中HSV-11例，占2．38%；HSV-241例，占97．62％。对于有皮损的患者，PCR法较酶标法有较高的阳性检出率。由此可见：(1)HSV-2是主要的致病因素；(2)在对病毒分型及有皮损患者的诊断上，PCR法明显优于酶标法。     

不典型生殖器疱疹临床分析

通过临床收集36例不典型GH,对其病史,症状,体征进行详细记录及随访,采用PCR检测局部棉拭子HSVⅡ－DNA和ELISA法检测血清抗HSV－Ⅱ的IgM确立诊断,同时检测相关病原体排除诊断,包括真菌,衣原体,支原体,梅毒,淋菌,滴虫等。结果：不典型GH主要表现为：1．生殖器部位细小溃疡;2．男性尿道外口炎症（尿道口炎）;3．生殖器非特异性红斑;4．龟头丘疹样小结节;5．非特异笥阴道炎,PCR检测HSVⅡ-DNA性23例（64％）,ELISA检测血清抗HSV－Ⅱ的IgM阳性36例（100％）。Ⅱ     

定量PCR和间接免疫荧光法联合检测生殖器疱疹病毒感染

目的：研究间接免疫荧光法（IIF）检测生殖器HSV感染的敏感性和特异性。方法：以定量PCR为对照,用HSV型共同性糖蛋白单克隆抗体为夹心的IIF法,检测了94例临床诊断为生殖器疱疹的患者皮疹中的HSV。结果：IIF法检测HSV的敏感性为74．12％,特异笥为55．60％;总阳性率（71．30％）,明显低于定量PCR法的阳性率（90．40％）（P＜0．05）,但两种方法检测GH患者皮水疱内的HSV阳性率无明显差异（86．20％vers.97.00%),而检测糜烂性皮疹内的HSV时,PCR法的阳性率高于IIF法（P＜0．05）,结论：IIF法具有简单,快捷的优点,适用于检测早期可疑GH患者皮疹内HSV,有临床实用价值。     

荧光定量聚合酶链反应在一期梅毒诊断中的临床意义探讨

为评价荧光定量聚合酶链反应（FQ－PCR）在一期梅毒诊断中的临床应用价值，以暗视野（D－F）、血清学（RPR／TPHA）检查方法作对照，用FQ－PCR方法检测68例疑诊为一期梅毒病人的生殖器溃疡处分泌物。在68例疑诊为一期梅毒病人中，D－F阳性率27．94％（19／68），RPR阳性率48．53％（33／68），TPHA阳性率61．76％（42／68），FQ－PCR阳性率44．12％（30／68），FQ－PCR与D－F、TPHA比较有显著性差异（P＜0．05），与RPR比较无显著性差异（P＞0．1）。本研究表明FQ－PCR检测生殖器溃疡TP在一期梅毒诊断中有一定的价值。     

生殖器标本单纯疱疹病毒抗原检测及其临床评价

为评价酶联免疫吸附试验（ELISA）检测生殖器疱疹病毒的临床应用情况，我们应用ELISA法检测645例生殖器标本中HSV抗原。ELISA法检测不同类型的皮损，HSV抗原检出率有差异，水疱、溃疡、结痂、丘疹红斑和裂隙的HSV抗原检出率分别为83．7％、66．3％、34．6％、12．9％和12．6％。ELISA法检测HSV感染，其敏感性和特异性强，具有方便、快速等特点，适合大批量样本的检测，值得临床推广使用，尤其在基层医院。     

荧光多重实时PCR检测单纯疱疹病毒

目的建立单纯疱疹病毒的荧光多重实时PCR检测及分型技术，为生殖器疱疹的诊断及病毒分型提供快捷、敏感、特异的方法。方法以细胞培养法为对照，用Smart Cycler System作为实验平台，以DNA探针荧光标记技术建立荧光多重实时PCR，用于检测生殖器疱疹患者临床标本中的1型和2型单纯疱疹病毒(HSV1、HSV2)DNA。结果荧光多重实时PXR检测HSV1和HSV2 DNA的敏感性和特异性均为100％，可检测出低至1～5拷贝的HSV DNA，30min左右即可完成整个检测过程．而细胞培养的敏感性和特异性分别为70．10％和100％。结论基于Smart Cycler System的荧光多重实时PCR是一种易于操作、扩增效率高、封闭式的核酸扩增技术，可用一次PCR反应就能快速、敏感、准确地对临床标本中的单纯疱疹病毒进行检测和分型。     

荧光多重PCR与血清型特异性抗体检测HSV感染的比较

目的：比较荧光多重PCR和血清型特异性抗体测定在生殖器疱疹临床诊断中的应用价值及评价各自的优缺点。方法：以细胞培养法作为“金标准”对照，分别用荧光多重PCR和血清型特异性抗体检测法对121例临床诊断为生殖器疱疹的标本进行检测。结果：以培养法作标准，并通过结果的差异性分析，荧光多重PCR的敏感性为100％，特异性为88．89％；血清型特异性抗体测定则分别为77．68％和77．78％，荧光多重PCR的敏感性和特异性均显高于血清型特异性抗体测（P＜0．05），但前不能检测出无皮损患HSV的DNA，而后可检测出无皮损患中的HSV抗体。结论：荧光多重PCR和血清型特异性抗体检测各有其自身的优缺点，单独用PCR和其它病毒分离的方法和单独使用血清特异性抗体检测的方法来诊断生殖器疱疹都是不完整的，均可造成漏诊。临床上将两种方法有机的结合起来应用能发挥各自的优势，取长补短，对早期、准确、快速地诊断生殖器疱疹及进行流行病学调查有着十分重要的意义和使用价值。     

Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic

BACKGROUND: Polymerase chain reaction (PCR) has well established advantages over culture for diagnosis of herpes viruses, but its technical complexity has limited its widespread application. However, recent methodological advances have rendered PCR more applicable to routine practice. Aim: To compare automated PCR with viral culture for diagnosis of genital herpes. METHODS: We studied 236 patients presenting with clinical features suggestive of genital herpes at an inner city genitourinary medicine clinic. Two swabs were taken from each patient. Cell culture and typing were performed by standard methods. Automated PCR was performed using the LightCycler instrument and the infecting viral type was determined by restriction endonuclease digestion of amplicons. RESULTS: 109 patients (46%) had a positive test for herpes simplex virus (HSV). In 88, both PCR and culture were positive; in 21 PCR only was positive. With both detection methods, lesion duration and morphology were associated with HSV detection. Compared with culture alone, use of PCR increased sensitivity by 13.3% in specimens from vesicular lesions, by 27.4% from ulcerative lesions, and by 20.0% from crusting lesions. CONCLUSIONS: We advocate adoption of automated PCR as an efficient HSV detection and typing method for diagnosis of genital herpes in routine clinical practice. PCR allowed rapid laboratory confirmation of the diagnosis and increased the overall HSV detection rate by 24%.     

生殖器部位皮损的单纯疱疹病毒检测及分型

目的 探讨生殖器疱疹部位皮损的不典型表现及其与单纯疱疹病毒型别的关系。方法 对外生殖器部位及其周围有硬结或疖肿、裂隙、毛囊炎等非水疱性皮肤黏膜损害的患者进行临床资料采集和分析,并对皮损标本进行单纯疱疹病毒的分离培养、PCR检测和病毒分型。结果 105例有外生殖器部位非水疱性皮损的患者入选本研究,在硬结(或疖肿)、裂隙、毛囊炎、类似擦破、单个溃疡、非特异性红斑和红肿渗液性包皮龟头炎皮损中,PCR检测HSV的阳性率分别33.3%(6/18)、20%(3/15)、37.5%(6/16)、28.6%(2/7)、33.3%(4/12)、20%(5/25)和50%(6/12),总的检出阳性率为30.5%(32/105)。分离培养法检测HSV的阳性率分别为22.2%(4/18)、13.3%(2/15)、25%(4/16)、14.3%(1/7)、33.3%(4/12)、8%(2/25)和41.7%(5/12),总的检出阳性率为21%(22/105)。两种方法检测HSV的总检出率差异无统计学意义(κ=0.095,P=0.114)。HSV-PCR分型结果与荧光单克隆抗体分型结果相符。在所有HSV阳性者中,HSV-1感染占9.4%(3/32),HSV-2感染占90.6%(29/32)。结论 生殖器HSV感染的皮肤黏膜损害多样,可为外生殖器部位的硬结(疖肿)、裂隙、毛囊炎、类似擦破、单个溃疡、非特异性红斑和红肿渗液性包皮龟头炎等不典型表现,而且主要由HSV-2感染引起。     

Increasing relative prevalence of HSV-2 infection among men with genital ulcers from a mining community in South Africa

OBJECTIVES: To determine the aetiology of genital ulcer disease (GUD) and its association with HIV infection in the mining community of Carletonville, South Africa, from two cross sectional surveys of consecutive men presenting with genital lesions during October 1993 to January 1994 and July to November 1998. METHODS: A multiplex polymerase chain reaction (M-PCR) assay combined with amplicon detection was used to identify DNA specific sequences of Treponema pallidum, herpes simplex virus (HSV), and Haemophilus ducreyi. A real time PCR assay was used to differentiate between HSV-1 and HSV-2. RESULTS: M-PCR detected T pallidum, HSV, and H ducreyi in 10.3%, 17.2%, and 69.4% of 232 GUD patients during 1993-4 and in 12.4%, 36.0%, and 50.5% of 186 GUD patients in 1998. The proportion of patients with more than one agent increased significantly from 7.3% (17/232) in 1993-4 to 16.7% (31/186) in 1998 (p <0.01). HSV-2 was detected in a higher proportion of ulcer specimens from HIV infected patients than in specimens from HIV uninfected patients during both time periods (1993-4: 26.2% v 6.7%, p <0.001; 1998: 42.1% v 29.6%, p >0.09). CONCLUSIONS: Based on two cross sectional surveys, 4 years apart, chancroid remained the leading cause of GUD in men who presented at the STD clinic with genital ulcers in the mining community of Carletonville, South Africa. The relative prevalence of primary syphilis has remained low. However, HSV-2 has emerged as a more significant cause of GUD and the proportion of GUD patients infected with more than one agent also increased significantly. HSV-2 DNA was detected in a significantly higher proportion of ulcer specimens from HIV positive patients than from HIV negative patients. No association was found between HIV infection status and the relative prevalence of chancroid or syphilis.     

间接免疫荧光法检测生殖器疱疹病毒

目的:评价间接免疫荧光试验(IFA)在生殖器疱疹(GH)诊断中的应用价值。方法:采用以单纯疱疹病毒(HSV)型共同性单克隆抗体为夹心的IFA法,检测了120例临床诊断为GH患者皮疹中的HSV,并与病毒培养法进行比较。结果:IFA检测HSV的总阳性率为85.8%,高于病毒培养法的阳性率(70.8%,χ2=12.04,P<0.01)。两种方法检测GH水疱内的HSV阳性率分别为93.3%和90.0%,无明显差异(χ2=1.96,P>0.05);而检测糜烂和结痂性皮疹内的HSV时,IFA的阳性率分别为92.6%和69.4%,均分别高于病毒培养法(75.9%,χ2=5.82,P<0.05;47.2%,χ2=14.17,P<0.01)。结论:IFA法具有简单、快速、敏感性高的优点,适于检测GH患者皮疹内HSV,有临床实用价值。     

生殖器疱疹病毒感染检测方法的临床应用

目的 :　评价检测生殖器疱疹病毒实验方法的临床应用价值。方法 :　同时采用细胞培养法、定量PCR法、间接免疫荧光法 (IIF)对 5 3例生殖器疱疹 (GH)感染患者进行了HSV检测。结果 :　细胞培养、定量PCR检测HSV阳性率明显高于IIF法 (P <0 .0 1) ,细胞培养与定量PCR比较无显著性差异 (P >0 .0 5 )。三种方法检测GH患者皮疹水疱内的阳性率无明显差异 (P >0 .0 5 )。IIF法检测糜烂结痂的阳性率仅为 2 8.6 %。结论 :　三种方法均适用GH水疱期患者标本的检查。而定量PCR、细胞培养检测GH糜烂结痂患者标本较IIF法敏感     

Diagnosis of genital herpes by real time PCR in routine clinical practice

BACKGROUND: Virus isolation in cell culture is the recognised diagnostic gold standard for genital herpes. Although increasing evidence indicates that polymerase chain reaction (PCR) provides a more rapid and sensitive diagnostic method, its implementation in routine diagnostic settings has been limited by concerns over contamination and cost. OBJECTIVE: To evaluate the feasibility of replacing virus culture with PCR for the diagnosis of genital herpes in settings serving large populations of genitourinary medicine (GUM) attendees. METHODS: Genital swabs collected from 233 consecutive GUM attendees with suspected genital herpes were tested in parallel by virus culture and automated real time PCR. Three specimen preparation methods were evaluated and the assay reliability was assessed by repeat testing, comparison with a commercially available assay, and herpes simplex virus (HSV) sequence analysis. Probe melting temperatures (Tm) were used to differentiate between HSV types without additional post-PCR steps. RESULTS: HSV was detected in 79/233 (34%) samples by virus culture and 132/233 (57%) samples by PCR. PCR significantly increased HSV detection in both early (< 5 days) and late (> or = 5 days) presentations and in both first and recurrent episodes. HSV detection and typing by PCR was achieved within less than 4 hours leading to a significant reduction in labour compared to virus culture. Most specimens (120/132, 91%) were typed as HSV-2. Results were highly reproducible. CONCLUSIONS: Real time PCR is a highly reproducible, rapid, and labour efficient method for HSV detection in genital swabs. Its implementation is feasible in routine diagnostic settings.     

Duplex real-time polymerase chain reaction assay for detection and quantification of herpes simplex virus type 1 and herpes simplex virus type 2 in genital and cutaneous lesions

BACKGROUND: A sensitive and specific method for detecting herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) is important for diagnosing genital and cutaneous infections. GOAL: The goal of this study was to compare quantitative real-time polymerase chain reaction (qPCR) with virus culture for diagnosis of genital and cutaneous HSV-1 and HSV-2. STUDY DESIGN: A duplex qPCR system for quantification of DNA from HSV-1 and HSV-2 was developed. Duplicate swabs for PCR and virus culture were collected from 89 patients attending our sexually transmitted infection and dermatology clinic. RESULTS: The duplex qPCR had a linear measure interval of 10-10 copies/mL. The detection limit was between 1 and 5 copies per reaction. qPCR detected HSV in 57 (64%) specimens and virus was isolated in 45 (50%) cases. First-episode infections showed higher viral quantities with a median value of 4.2 x 10 copies per reaction compared with recurrent infections with 1.0 x 10 (P = 0.0002). HSV-1 was more likely to be the cause of first-episode genital infections (72%), and HSV-2 of recurrent and atypical genital manifestations (73%). CONCLUSION: Real-time PCR is a sensitive method for diagnosing genital herpes, and the duplex format is convenient for typing. The method increased the detection rate by 27% compared with virus culture.     

抗原捕获聚合酶链反应分型检测妇女生殖器单纯疱疹病毒

目的 :　建立直接分型检测妇女生殖器单纯疱疹病毒 (HSV)的抗原捕获聚合酶链反应 (AC -PCR)。方法 :　用抗HSV型共同性糖蛋白单克隆抗体 ,包被聚苯乙烯离心管 ,捕获HSV ,同时加入 3个引物 :HSV - 1 HSV - 2型共同性上游引物及HSV - 1和HSV - 2型特异性下游引物 ,进行PCR扩增。结果 :HSV - 1和HSV - 2标准病毒株均分别扩增出与设计大小相符的 4 77bp和 399bpDNA条带。AC -PCR可检测到 10PFUHSV - 1和 1PFUHSV - 2。用AC -PCR检测了 36 5份妇女生殖器拭子标本 ,阳性 10 1例(2 7.7% ) ,2 3例为HSV - 1(占 2 2 .8% ) ,78例为HSV - 2 (占 77.2 % ) ;其中 112份标本同时用AC -PCR和分离培养法检测 ,AC -PCR的阳性率为 2 6 .8% (30 112 ) ,分离培养法的阳性率为 2 0 .5 % (2 3 112 ) ,两者差异有显著性 (χ2 =4 .5 ,P <0 .0 5 )。结论 :　AC -PCR是特异、敏感、快速分型检测妇女生殖器HSV感染的方法