4212例泌尿生殖道感染沙眼衣原体PCR检测研究

４２１２ 例泌尿生殖道感染患者沙眼衣原体（ＣＴ） 行ＰＣＲ 检测，总阳性率２２－７９％ ；ＣＴ 阳性者中，合并１３ 项其他（ＮＧＨ、ＵＵ、ＨＰＶ）感染的占６８－７５％ ，ＣＴ 感染率男性（２２－８６ ％） 与女性（２２－７６％ ）十分相近，但合并感染率女性（７１－４３ ％ ）显著高于男性（６２－５ ％） ，Ｐ＜ ０－０１。本组结果显示，ＣＴ 主要通过性接触传染。     

用PCR—SSCP检测母婴标本中沙眼衣原体的研究

用配对母胎标本探讨沙眼衣原体的垂直传播。材料与方法；用细胞培养，聚合酶链反应和单链构象多态性检查配对母胎标本中的沙眼衣原体。结果；３０份宫颈标本中８份沙眼衣原体培养和质粒ＰＣＲ均为阳性；９名宫颈标本质粒ＰＣＲ阳性的孕妇中有４份羊水标本培养和ＰＣＲ均为阳性；１例新生儿睑结膜和配对羊水标本表现相同的ＳＳＣＰ图谱。     

套式PCR法检测输卵管妊娠者的沙眼衣原体感染研究

目的：为探索输卵管妊娠与生殖道沙眼衣原体的关系。方法：采集５７例输卵管妊娠者的宫颈分泌物、输卵管组织、胚胎组织、并采集３２例卵巢囊肿者的宫颈分泌物、输卵管组织和２９例早孕人流者的宫颈分泌物、胚胎组织。应用套式ＰＣＲ法检测以上标本中的沙肯衣原体ＤＮＡ。结果：输卵管妊娠者的宫颈分泌物阳性率高达４０．４％，明显高于卵巢囊肿者的１２．５％和早孕人流者的１３．７％，ＲＲ为４．４９；输卵这妊娠者的输卵管组织阳     

沙眼衣原体感染的PCR诊断和分型

沙眼衣原体(CT)是一种专性细胞内寄生苗，可引起宫颈、尿道、直肠、鼻咽和结膜等部位的粘膜感染。宫颈感染可逆行至宫内膜和输卵管从而引起盆腔炎、肝周围炎、不育症和异位妊娠。妊娠期感染可影响新生儿，造成新生儿结膜炎和婴儿肺炎。在男性，最常见的性传播疾病之一——非淋菌性尿道炎约40—50%为CT感染所致，CT并可引起同性恋者的直肠炎．除泌尿生殖道感染外，CT是沙眼的病原体，沙眼仍是发展中国家的致盲病因之一。     

新生儿疟眼沙眼衣原体和解脲脲原体感染的PCR检测研究

为探讨沙眼衣原体（ＣＴ）和解脲解原体（ＵＵ）与新生儿疾病的关系，我们采用套式聚合酶链反应（ＰＣＲ）技术，从１００例住院新生儿的结膜拭子中共检出阳性者５８例阳性率５８％其中ＣＴ３６例，ＵＵ２２例）；剖宫产７例（ＣＴ４例，ＵＵ３例）经产道分娩５１例（ＣＴ３２例，ＵＵ１９例）结果表明：新生儿是ＣＴ，ＵＵ易感者，可引起新生儿肺炎，宫内感染，早产，低体重，结膜炎，肠炎，采用先进的套式ＰＣＲ技术检测具有灵敏，     

应用PCR方法检测孕妇血中HCMV感染

聚合酶链反应(PCR)是新发展起来的分子生物学领域中的一项检测技术。我们应用PCR扩增技术，对妇产科临床怀疑为HCMV病毒感染的22～38岁的孕妇160例进行检测，其中阳性68例，阴性92例，阳性率42．5％。     

新生儿肺炎患儿沙眼衣原体PCR检测研究

应用聚合酶链反应（ＰＣＲ）检测沙眼衣原体（ＣＴ）ＤＮＡ，８４例新生儿肺炎患儿的咽拭子中共检出２１例，阳性率２５．Ｏ％。其中剖宫产儿阳性率１６．６％（２／１２）；经产道产儿阳性率２６．４％（１９／７２）；早产、低体重儿阳性率５０％（５／１０）；而对照组２０例健康新生儿无１例检出。提示新生儿是ＣＴ感染的高危群体；其感染ＣＴ以通过孕妇产道获得为主，但也有宫内感染；早产、低体重儿最易被ＣＴ感染，ＰＣＲ检测ＣＴ感染具有快速、敏感、特异的优点，有广阔的应用前景。     

孕早期沙眼衣原体宫内感染的检测

本研究利用ＰＣＲ技术和地高辛标记的ＤＮＡ探针对５９例早孕绒毛组织中沙眼衣原体染进行研究。结果发现２例绒毛组织有沙眼衣原体感染，并感染率为３．４％，研究证实沙眼衣原体可引起孕早基歼宫内感染，建议对沙眼衣原体感染的孕妇进行宫内感染的产前诊断。     

妊娠期妇女沙眼衣原体感染的检测报告

妊娠期妇女的各种衣原体感染常可引起自然流产、早产、胎膜早破、足月低体重儿及新生儿发病率的增加。性生活活跃期的妇女中约２０％－４０％有衣原体接触史，本室采用ＰＣＲ检测法对早、中期妊娠妇女沙眼衣原体感染进行了检测研究。资料与方法１．临床资料：９７年元月至...     

实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。     

Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay.

To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR.     

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay.

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.     

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.     

Detection of Mycobacterium leprae infection by PCR.

PCR amplification of the 531-bp fragment of the Mycobacterium leprae pra gene in fresh biopsy and slit skin smear samples was evaluated for its usefulness in the detection of leprosy bacilli in patients in Thailand. In multibacillary patients, 87.1% (27 of 31) of biopsy specimens and 41.9% (13 of 31) of slit skin smear specimens were positive by PCR, whereas in paucibacillary patients, 36.4% (8 of 22) of biopsy specimens and 18.2% (4 of 22) of slit skin smear specimens yielded detectable PCR amplification. Compared with other diagnostic procedures, PCR showed a clear advantage over both microscopic examination of slit skin smears and serologic detection of anti-phenolic glycolipid 1 antibody, especially in paucibacillary patients when bacterial indexes were 0 and seropositivity was only 6.25%. PCR was also evaluated for its potential to help monitor bacterial clearance in some of these patients during chemotherapeutic treatment. The PCR results on slit skin smear samples at 1, 3, and 6 months of chemotherapy showed that the number of PCR-positive cases of both multibacillary and paucibacillary types decreased sequentially. The results of this study are encouraging. However, investigation of a larger number of clinical specimens with an improvement in PCR methods, especially on slit skin smears, needs to be done before PCR can be established as a diagnostic procedure for leprosy patients and subclinical cases or as a tool for drug assessment.     

Diagnosis of Chlamydia trachomatis infections in asymptomatic men and women by PCR assay.

A PCR assay was evaluated for its ability to detect genital chlamydial infection in asymptomatic men and women. Urethral swab specimens were collected from 472 men for culture and PCR assay, and first-void urine (FVU) specimens were collected from 379 of these men for enzyme immunoassay (EIA) and PCR assay. Cervical swab specimens were collected from 242 women for culture, EIA, and PCR assay. Patients were considered infected if they were culture positive or positive by PCR with both plasmid- and major outer membrane protein-based primers. By using this extended "gold standard," the prevalence of infection in this population was 7.6% for men and 7.9% for women. For men, the sensitivities of urethral swab specimen culture and PCR and FVU specimen EIA and PCR were 61, 72, 55, and 91%, respectively. All assays had specificities of > or = 99.8%. The positive and negative predictive values for PCR testing of FVU specimens were 100 and 99.4%, respectively, compared with values of 96.3 and 97.8%, respectively, for PCR of urethral swab specimens. The sensitivities of cervical swab specimen culture and PCR testing were 42 and 90%, respectively, with corresponding specificities of 100 and 99.3%. All cervical swabs were negative by EIA. Molecular techniques such as PCR assays are valuable tools for the detection of symptomatic genital chlamydial infection. In particular, PCR assays of FVU specimens from men offer a highly sensitive, noninvasive screening tool that will likely improve patient compliance for diagnostic testing.     

荧光定量PCR检测沙眼衣原体及临床应用

目的：用荧光定量聚合酶链反应（FQ－PCR）检测沙眼衣原体的含量,探讨FQ－PCR在衣原体性尿道炎诊断中的价值。方法：应用荧光探针标记引物的荧光定量聚合酶链反应对172例疑为沙眼衣原体感染患者标本进行检测,并与常规PCR（电泳－EB染色）进行比较。结果：FQ－PCR阳性率为19．8％,常规PCR法为20．9％,两法符合率为63．9％。女性宫颈分泌物标本FQ－PCR阳性为29．2％,常规PCR法为16．7％;男性分泌物FQ－PCR阳性率为16．9％,常规PCR法为13．8％。FQ－PCR特异性较常规PCR法高。结论;FQ－PCR在扩增中实时在线检测,并选择理想的标准曲线做出较精确的临值分析,具有较高的特异性和敏感性,对病原体的诊断有一定的临床意义。     

Detection and genotyping of human papillomavirus by real-time PCR assay

BackgroundDiagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients.ObjectiveOur study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples.Study designValidation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity.ResultsData showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10³ cells using in-house HPV (6, 11 and 16) viral load assays.ConclusionThe real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.