沙眼衣原体荧光定量PCR检测方法的建立

目的建立沙眼衣原体实时荧光定量PCR检测技术,以期用于沙眼衣原体的感染检测。方法针对沙眼衣原体ompA基因序列保守区域设计引物和TaqMan探针,构建标准质粒并制作标准曲线,建立沙眼衣原体实时荧光定量检测方法,通过对人型支原体等的检测评价方法的特异性。结果建立的沙眼衣原体荧光定量PCR体系能特异性检测沙眼衣原体,与人型支原体、解脲脲原体、淋球菌、大肠埃希菌EDL933、金黄色葡萄球菌无交叉反应;标准曲线在3.9×109～3.9×103 copies/μl之间线性关系良好(r2=0.99);方法的灵敏度高,检测最低拷贝数为3.9copies/μl;组内及组间变异系数均5%。结论建立的检测沙眼衣原体实时定量PCR方法特异、灵敏,可用于沙眼衣原体感染的早期筛查和临床快速诊断。     

检测单纯疱疹病毒2型的实时荧光定量聚合酶链反应方法的建立

目的建立实时荧光定量聚合酶链反应(real-time PCR)方法,检测单纯疱疹病毒2型(HSV-2)。方法根据基因库信息,以HSV-2的gG基因区为靶基因,设计合成引物和荧光探针。采用重组质粒构建标准品,优化反应体系进行方法学评价,同时对350例临床诊断HSV-2感染者和50例健康体检者的分泌物进行检测。结果成功建立了TaqMan技术的real-time PCR方法。通过反应体系优化重复性良好,批内变异系数CV=2.56%,批间CV=5.15%。特异性高,对乙型肝炎病毒、丙型肝炎病毒及解脲支原体、沙眼衣原体阳性标本进行检测无特异性扩增,灵敏度达到500拷贝。从350例临床诊断HSV-2感染者的分泌物中,检出HSV-2DNA 288例,检出率82.29%;50例健康体检者分泌物中未检出HSV-2DNA。结论 real-time PCR方法能够快速、准确地检出HSV-2DNA,其特异性强、灵敏度高,可用于临床诊断HSV-2感染。     

鹦鹉热嗜衣原体实时定量PCR检测方法的建立

目的建立一种简便、快速、特异的荧光定量PCR检测方法,用于鹦鹉热嗜衣原体的快速检测。方法以主要外膜蛋白(MOMP)基因为靶序列设计特异性引物,采用SYBR GreenⅠ随机渗入法建立实时定量PCR检测方法。结果循环阈值(Ct)与标准DNA模板在1.0×102-1.0×107拷贝/μl浓度范围内呈良好的线性关系,相关系数为0.989。该方法用于鹦鹉热嗜衣原体的检测具有很高的特异性,其敏感性与常规PCR相比可以提高100倍。结论本研究建立的检测鹦鹉热嗜衣原体的实时定量PCR检测方法具有很高的特异性和敏感性,可以用于鹦鹉热嗜衣原体的检测。     

沙眼衣原体感染实验室诊断研究

目的 探讨几种检测泌尿生殖道沙眼衣原体感染方法的临床诊断价值。方法 用细胞培养法、糖原试验、PCR和透明窗试验分别对81例STD门诊女性患者宫颈分泌物标本进行平行检测。结果 以细胞培养作参比，糖原试验的敏感性为87.5%，特异性为98.6%；PCR敏感性为1005，特异性为98.6%；透明窗试验敏感性为75.0%，特异性100%。结论四种检测方法均可应用于沙眼衣原体感染的诊断，且糖原试验是一种快速、易行、经济的方法。     

一种基于momp基因的衣原体分子生物学甄别技术的初步建立

目的利用PCR方法建立一种基于momp基因的衣原体分子甄别方法。方法根据衣原体momp基因的恒定区和可变区分别设计衣原体科特异性引物和种特异性引物，利用PCR方法对本实验室保存的衣原体进行扩增，以达到甄别的目的。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，研究momp基因的多态性。结果利用建立的PCR方法对本实验室保存的九株衣原体进行了研究，结果表明八株衣原体都是鹦鹉热嗜衣原体，一株为沙眼衣原体。利用限制性片段长度多态性（RFLP）分析科特异性PCR扩增产物，分析了D34、B11001、CW2和CP12四株衣原体，结果可以产生两种不同的带型。结论利用建立的PCR方法可以达到检测甄别衣原体的目的，并可以通过RFLP分析衣原体的侵袭性。     

性病门诊人群泌尿生殖道沙眼衣原体混合感染及分型研究

目的 了解性病门诊人群泌尿生殖道沙眼衣原体混合感染及基因型分布特点。方法 对2048例性病门诊就诊者分别取尿道或宫颈拭子，实时聚合酶链反应（PCR）检测沙眼衣原体（CT）感染，阳性标本用套式PCR扩增ompl VD2区片断，反向线点杂交（RLB）检测特异性型别。结果 CT总体感染率为15．9％（325／2048）。对287份阳性标本成功分型，型别分布为：F21．6％，E20．9％，J19．9％，D12．9％，G8．4％，K4．2％和H1．4％，31份（10．8％）为混合感染。结论 在性病门诊人群中CT感染率较高，主要流行型别为F、E和J型，且具有较高的混合感染率。     

肺炎衣原体PCR、套式PCR评价与临床应用

目的评价PCR和套式PCR检测肺炎衣原体(Chlamydia pneumoniae,Cpn)的特异性和灵敏度。方法按Campbell等建立PCR反应体系,按Tong等与秦玲等建立套式PCR反应体系,本文并以Cpn 16SrRNA基因(16SrDNA)为靶基因自行建立套式PCR反应体系,对Cpn、沙眼衣原体、鹦鹉热衣原体、大肠埃希稀菌、肺炎克雷伯菌、流感嗜血杆菌、粘质沙雷菌、洛菲不动杆菌、鲍曼不动杆菌、嗜肺军团杆菌、巨细胞病毒、单纯疱疹病毒进行检测以评价各方法的特殊性异性;以纯化的Cpn-DNA和1例阳性临床标本作梯度稀释后分别参与各反应体系检测以评价各方法的灵敏度。结果1、PCR和套式PCR均只能检出Cpn TW-183株和CWL-29株,而其它衣原体、支原体、细菌、病毒均不能检出;2、套式PCR检测Cpn较PCR敏感100～1000倍;3、经以16SrRNA基因为靶基因套式PCR检测新生儿肺炎(非吸入性肺炎)和儿童肺炎者咽拭子标本Cpn阳性率分别为16.2％和22.0％,肺结核者痰标本为18.9％,男性STD者尿道拭子标本为0％。结论 PCR和套式PCR检测Cpn均有较高的特异性,其中套式PCR比PCR敏感100～1000倍,在肺部炎症者咽拭子或痰标本中Cpn有较高的检出率。     

福州市性病门诊人群生殖道沙眼衣原体的分子流行病学研究

目的 探讨福州市性病门诊人群泌尿生殖道沙眼衣原体的基因型分布状况。方法 收集2013-2014年临床疑似生殖道沙眼衣原体感染者标本2 019份,用实时荧光定量PCR检测沙眼衣原体,阳性标本用套式PCR扩增Omp1区并进行测序分析。测序结果上传至BLAST网站查找序列的相似性、构建系统树确定基因型。结果 检测沙眼衣原体阳性标本86份,成功分型的有83份,1株为混合感染。测得8个基因型,分别为:F型(36.59%)、E型(24.39%)、D型(12.20%)、G型(9.76%)、J型(9.76%)、H型(3.66%)、K型(2.44%)、B型(1.22%)。计算不同基因型间Omp1基因的核苷酸的同义突变率(d_S)和非同义突变率(d_N),发现仅G型的d_N/d_S值大于1,其他型均小于1。结论 F型和E型是福州市性病门诊人群泌尿生殖道沙眼衣原体的主要基因型。同义替代是Omp1基因在进化过程中的主要变异。     

二聚体蝎型探针荧光定量PCR快速检测志贺菌方法的建立及初步应用

目的 利用二聚体蝎型荧光探针法,建立一种可广泛应用且敏感、特异的检测志贺菌的荧光定量PCR技术。方法 根据编码志贺菌ipaH基因的核苷酸序列设计引物和荧光探针,采用基因重组技术构建用于志贺菌检测的定量标准品,通过对荧光定量PCR反应体系和反应条件的摸索,建立了检测志贺菌的二聚体蝎型探针定量PCR方法。结果 成功构建了志贺菌重组质粒标准品和志贺菌实时荧光定量PCR方法;通过特异性、敏感性、稳定性和重复性验证,结果表明具有较好的特异性、敏感性、稳定性和重复性;对比分离培养标本,二者符合率为100％;对比TaqMan方法,本方法具有更敏感、省时、成本低的特点。结论 建立了一种以二聚体蝎型荧光探针技术为基础的志贺菌荧光定量PCR检测法,为研制新型的检测试剂盒奠定了基础,可应用于食品卫生监管以及传染病诊断等。     

F型沙眼衣原体主外膜蛋白基因omp1原核表达及其序列分析

目的对F型沙眼衣原体主外膜蛋白(MOMP)基因进行原核表达,获得omp1基因重组蛋白,并对克隆到的基因进行序列分析,为F型沙眼衣原体的诊断及疫苗研究奠定基础。方法利用PCR技术从沙眼衣原体阳性病人尿道拭子标本中扩增omp1基因,分析序列,并定向克隆入原核表达载体pGEX-5X3中,与GST进行融合表达。结果对克隆到的沙眼衣原体omp1基因进行序列分析,基因序列与F型沙眼衣原体F/IC-CAL3相似性达99%,构建Omp1-pGEX-5X3重组质粒,omp1与GST融合表达。结论成功构建F型沙眼衣原体omp1基因原核表达系统,得到重组蛋白,为沙眼衣原体疫苗的研究和临床检测试剂盒的研制打下基础。     

Usefulness of real-time PCR in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical swabs and first-voided urine specimens

We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detecting N. gonorrhoeae was 100% in all 3 specimentypes. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.     

Evaluation of the new nucleic acid amplification system for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in women

The performance of a real-time DNA amplification assay, BD ProbeTec ET System (BDPT, BD Diagnostic Systems), to detect Chlamydia trachomatis and Neisseria gonorrhoeae on endocervical and oropharyngeal samples was evaluated. After obtaining informed consent, 364 endocervical, 363 urine and 247 oropharyngeal specimens were collected from 307 cases. The overall agreement rate of the BDPT and Amplicor (AMP, Roche) assays for the detection of C. trachomatis and N. gonorrhoeae in endocervical samples was 99.2% (361/364) for C. trachomatis and 99.5% (362/364) for N. gonorrhoeae. Assay of oropharyngeal swabs by the BDPT yielded 21 C. trachomatis positives, and 19 of them were C. trachomatis negative by the DNA probe assay (Gen-Probe PACE). The AMP assay showed that 16/19 (84.2%) of the BDPT +/DNA probe - samples were positive. The BDPT also yielded 21 N. gonorrhoeae positives, 15 of which were negative with the DNA probe. Additional testing showed that all 15 BDPT +/DNA probe - samples were positive by the established nested PCR method. Our data suggest that the performance of the BDPT is comparable to that of AMP for detection of C. trachomatis and N. gonorrhoeae in endocervical swab samples and that it may be a useful method for detecting of C. trachomatis and N. gonorrhoeae in oropharyngeal samples clinically.     

泌尿生殖道标本中沙眼衣原体的直接检测及基因分型

应用聚合酶链反应（PCR）和限制性片段长度多态性（RFLP）分析，建立了沙眼衣原体（Ct）直接检测及基因分型的方法。与细胞培养法比较，评价其检测的敏感性。400份性病门诊患者的泌尿生殖道标本同时用培养和质粒PCR检测Ct，结果29份标本培养阳性，其中28份标本质粒PCR阳性，另有5份培养阴性的标本质粒PCR阳性。对培养和质粒PCR阳性的标本进一步做主要外膜蛋白基因（ompl）PCR或ompl巢式PCR，结果32份标本omplPCR或ompl巢式PCR阳性。将阳性标本的ompl产物用AluI和MspI等限制性内切酶酶切，产生的酶切带谱与标准株带谱比较，以鉴定阳性标本的基因型。结果E型为13份标本，F：6、G：4、D：3、J：2、K：2、H：1、非典型：1。研究表明：PCR－RFLP可直接检测及基因分型泌尿生殖道标本中的Ct，为一种敏感的血清分型替代方法。     

Comparison of polymerase chain reaction and enzyme immunoassay (Chlamydiazyme) in detection of Chlamydia trachomatis from male urethritis]

A method using polymerase chain reaction (PCR) was compared to an enzyme immunoassay (Chlamydiazyme) for detection of Chlamydia trachomatis by testing a reference strain and clinical specimens. Two oligonucleotides based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used as primers for the PCR. A DNA fragment of 242 bp specific for C. trachomatis was amplified by the PCR, when DNA of greater than or equal to 10(2) C. trachomatis was used as template for the PCR. A chlamydial antigen was detected by Chlamydiazyme, when greater than or equal to 2.6 x 10(3) C. trachomatis were applied for the enzyme immunoassay. The PCR method was 26 times more sensitive than Chlamydiazyme in detection of C. trachomatis. The PCR method and Chlamydiazyme were carried out to examine 74 urethral swabs obtained from male patients with urethritis for detection of C. trachomatis. In 45 of 74 specimens, the DNA fragment of C. trachomatis was amplified by the PCR, and in 41 of 74, the chlamydial antigen was detected by Chlamydiazyme. The detection rate of the PCR method (60.8%) was higher than that of Chlamydiazyme (55.4%). The positive coincidence rate of the PCR method to Chlamydiazyme was 100% (41/41) and negative coincidence rate was 87.9% (29/33). The overall coincidence rate between the two methods was high (94.6%). Thus, the PCR method was more sensitive than Chlamydiazyme for detection of C. trachomatis and specific for diagnosis of chlamydial urethritis.     

基于SYBR Green检测微小隐孢子虫的实时荧光定量PCR方法的建立及应用

目的建立一种方便快捷的检测微小隐孢子虫的实时荧光定量PCR（Real-time PCR）方法。方法根据GenBank公布的微小隐孢子虫18s rRNA基因序列设计1对引物,通过质粒DNA和卵囊检测标准曲线的绘制及特异性和重复性的研究,建立检测微小隐孢子虫的基于SYBR Green的Real-time PCR方法,进而对奶牛粪便进行检测。结果建立的Real-time PCR法对检测微小隐孢子虫具有很高的特异性,质粒DNA和卵囊的检测阈值分别达到1个拷贝和10个卵囊,奶牛粪便阳性率为25.93%（21/81）,高于普通PCR检测率20.99%（17/80）。结论建立的Real-timePCR方法简便、快速,特异性强,敏感度高,可用于微小隐孢子虫的快速定量检测。     

Detection of Chlamydia trachomatis from urethral swab in male urethritis by polymerase chain reaction]

A polymerase chain reaction (PCR) with Chlamydia trachomatis-specific primers was applied for detection of C. trachomatis from urethral swab in male urethritis. The results were compared with those of culture method for detection of C. trachomatis. Of 18 clinical specimens tested in this study, inclusion bodies of C. trachomatis were detected in 11 specimens by the culture method. For PCR, sample DNA was prepared from transport medium in which urethral smear was suspended and two oligonucleotides based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used as extension primers. In 12 of the 18 specimens, 242bp DNA fragment was amplified by PCR and demonstrated to be the DNA fragment of C. trachomatis by Southern blot hybridization. No DNA of 242bp was amplified by PCR from five specimens in which any inclusion bodies of C. trachomatis were observed or from a specimen in which one inclusion body per cover slip was detected by culture method. C. trachomatis DNA of 242bp was amplified from all specimens in which 14 and more inclusion bodies per cover slip were detected by culture method. In two specimens concluded s negative by culture method, amplified C. trachomatis DNA were detected by PCR. Thus, the PCR would be a more simple and sensitive method for detection of C. trachomatis, compared with the culture method.     

Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction

OBJECTIVE: Molecular biology techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) are routinely used in research for detection of C trachomatis DNA in synovial samples, and these methods are now in use in some clinical laboratories. This study aimed at determining the method best suited to molecular diagnosis of C trachomatis by examining four standard DNA preparation methods using chlamydia spiked synovial tissue and chlamydia infected monocytes. METHODS: Synovial tissue from a chlamydia negative patient with rheumatoid arthritis was spiked with defined numbers of C trachomatis elementary bodies (EB). Purified human peripheral monocytes from normal donors were infected with the organism at a multiplicity of infection 1:1 in vitro and harvested after four days. DNA was prepared from all samples by four methods: (1) QIAmp tissue kit; (2) homogenisation in 65 degrees C phenol; (3) incubation at 97 degrees C; (4) proteinase K digestion at 97 degrees C. DNA from methods 1 and 2 was subjected to PCR using two different primer sets, each targeting the C trachomatis omp1 gene. LCR was done on DNA prepared by each method. RESULTS: In synovial tissue samples spiked with EB, and in monocytes persistently infected with the organism, preparation of template using the QIAmp tissue kit (method 1) and the hot phenol extraction technique (method 2) allowed sensitive detection of C trachomatis DNA. These methods also produced template from both sample types for LCR. DNA prepared by heat denaturation (method 3) allowed only low sensitivity chlamydia detection in LCR and did not work at all for PCR. Proteinase K digestion plus heat denaturation (method 4) gave template that did not allow amplification in either PCR or LCR assays. CONCLUSIONS: The sensitivity of detection for C trachomatis DNA in synovial tissue by PCR and LCR depends strongly on the method used for preparation of the amplification template. LCR targeting the multicopy chlamydial plasmid and two nested PCR assay systems targeting the single copy omp1 gene showed roughly equivalent sensitivity. Importantly, template preparation method and the specific PCR primer system used for screening must be optimised in relation to one another for highest sensitivity.     

Detection of Chlamydia trachomatis by polymerase chain reaction]

A polymerase chain reaction (PCR) procedure was developed for detection of Chlamydia trachomatis. Two oligonucleotide primers based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used. A single DNA fragment was amplified, when C. trachomatis DNA was template for the PCR. No amplified product was detected in Chlamydia psittaci DNA, Chlamydia pneumoniae DNA or other bacterial DNAs. The amplified DNA fragment was detected, when DNA of greater than or equal to 10(2) C. trachomatis per reaction was used as template for the PCR. Thus, the PCR was shown to be specific for C. trachomatis and more sensitive than the enzyme immunoassays for detection of chlamydial antigen and the chlamydial rRNA:DNA probe hybridization method.