聚合酶链反应-单链构象多态性检测淋病奈瑟菌gyrA基因突变的研究

国外研究表明 ,DNA旋转酶是淋病奈瑟菌 (淋菌 )中氟喹诺酮类药物的首要作用靶位 ,淋菌对氟喹诺酮类药物耐药主要与编码该酶的gyrA基因喹诺酮耐药决定区 (QRDR)突变有关[1] 。为建立一种简便、快速、可靠的淋菌gyrA基因突变的检测方法 ,并进一步探讨该突变与我国临床分离淋菌对氟喹诺酮类药物耐药的关系 ,本课题以浓度梯度 (Etest)法检测了 42株临床分离淋菌对环丙沙星敏感性 ,采用聚合酶链反应 单链构象多态性分析 (PCR SSCP)结合DNA测序技术 ,对淋菌gyrA基因QRDR突变进行了研究 ,报告如下。一、材料与方法1.菌株来源 :本研究中 4…     

加德纳菌实验诊断方法的评价和临床意义

目的探讨加德纳氏菌实验室检测方法的应用效果,为细菌性阴道病的诊断和治疗提供切实可行的诊断依据.方法应用革兰氏染色、吖啶橙染色、分离培养鉴定、聚合酶链反应检测阴道或宫颈分泌物中的加德纳氏菌.结果经在50例宫颈分泌物标本中,革兰氏染色阳性检出率为18.0%(9/50)、吖啶橙染色阳性检出率为20%(10/50)、分离培养阳性检出率为16.0%(8/50),聚合酶链反应阳性率为28.0%(14/50).结论通过革兰氏染色、吖啶橙染色、分离培养鉴定、聚合酶链反应检测结果显示,用聚合酶链反应检测阴道或宫颈分泌物中的加德纳氏菌用于细菌性阴道病的快速诊断,明显优于三种常规的检测方法.     

培养结合聚合酶链反应检测解脲脲原体

解脲脲原体（Ｕｕ）感染的发病率有日渐增高的趋势［１］。分离培养检测Ｕｕ操作繁琐、周期长。聚合酶链反应（ＰＣＲ）受诸多因素影响且系统本身的局限性难以避免。为提高准确率、缩短检测周期,我们采用培养结合聚合酶链反应检测２２１例泌尿生殖道感染患者Ｕｕ,现将结...     

妇女生殖道感染的PCR检测分析和护理

作者於1995年3月开始对本院门诊妇科接诊的238名患者,用PCR(聚合酶链反应)技术分别用患者阴道分泌物进行淋菌、沙眼衣原体、解脲支原体的检测。发现淋菌阳性率13.87％(33例)、沙眼衣原体阳性率10％(24例)、解腺支原体13.87％(33例),反映病原体感染和性传播性疾病感染在妇女生殖道感染占有一定比例。通过PCR技术快速、准确诊断,为患者及时治疗,对提高门诊诊治质量水平具有重要意义。     

荧光定量聚合酶链反应和液体培养法检测解脲支原体感染的临床意义

目的:探讨荧光定量聚合酶链反应和液体培养法检测解脲支原体感染的临床意义。方法:用荧光定量聚合酶链反应和液体培养法检测生殖道感染患者216例,无临床症状体检者84例解脲支原体,及确诊解脲支原体感染患者治疗后解脲支原体,比较2种方法检测阳性率。结果:治疗前、后荧光定量聚合酶链反应阳性检出率均高于液体培养法(P<0.01),但治疗后拷贝数明显下降。结论:临床应根据荧光定量聚合酶链反应及液体培养结果,结合临床综合判断,避免液体培养法的低灵敏度造成漏诊,延误治疗或荧光定量聚合酶链反应的高灵敏度造成误诊。     

聚合酶链反应鉴定艰难梭菌产毒株

以艰难梭菌Ａ毒素基因非重复片段中的２个寡核苷酸作引物，扩增３０６ｂｐ，用多聚酶链反应技术扩增３１株细菌，结果１９株艰难梭菌产毒株均扩增出单一特异的电泳带，面８株艰难梭菌无毒株，２株索氏梭菌和２株大肠杆菌均无特异带出现，将艰难梭菌产毒株的模板ＤＮＡ从５０ｎｇ稀释至０．５ｎｇ后再进行聚合酶链反应，结果仍可见行异扩增带，说明聚合酶链反应鉴定艰难梭菌产毒株较之细菌分离培养，细胞毒素测定方法具有快速。简便，     

从肺炎患者的痰分离出一株嗜肺军团菌5型

从丙型肝炎早期肝硬化感染肺炎的患者的痰中分离出１株军团菌，分离株的培养特性、形态染色和生物学特征、电镜观察所见均符合军团菌的特点，气相色谱分析表明，含有军团菌特异组分－－异十五烷酸，利用嗜肺军团菌特异引物对分离菌染色体ＤＮＡ进行聚合酶链反应，得到嗜肺军团菌特有的７８ｂｐ片断，血清学分型为５型。     

从肺炎患者的痰分离出一株嗜肺军团菌5型

从丙型肝炎早期肝硬化感染肺炎的患者的痰中分离出１株军团菌，分离株的培养特性、形态染色和生物学特征、电镜观察所见均符合军团菌的特点，气相色谱分析表明，含有军团菌特异组分──口异十五烷酸，利用嗜肺军团菌特异引物对分离菌染色体ＤＮＡ进行聚合酶链反应，得到嗜肺军团菌特有的７８０ｂｐ片断，血清学分型为５型。     

聚合酶链反应检测淋球菌的临床应用

报告应用聚合酶链反应(Polymorase chain reaction PCR)方法对89名性罪错青年女性采取宫颈分泌物进行淋球菌检测,同时用直接涂片和分离培养淋球菌,阳性率分别为51.69％、7.87％和16.85％。PCR法对淋球菌的检出率均较直接涂片法和分离培养法为高,其中15名分离培养阳性者,其PCR法也为阳性。笔者认为PCR法直接检测临床标本淋球菌具有特异、敏感、快速和简便的优点,该法无论对淋球菌检出率的提高和控制淋病的社会传播都有重要的意义。     

聚合酶链反应鉴定艰难梭菌产毒株

以艰难梭菌Ａ毒素基因非重复片段中的２个寡核苷酸链作引物，扩增３０６ｂｐ，用多聚酶链反应技术扩增３１株细菌，结果１９株艰难梭菌产毒株均扩增出单一特异的电泳带，而８株艰难梭菌无毒株、２株索氏梭菌和２株大肠杆菌均无特异带出现。将艰难梭菌产毒株的模板ＤＮＡ从５０ｎｇ稀释至０．５ｎｇ后再进行聚合酶链反应，结果仍可见特异扩增带。说明聚合酶链反应鉴定艰难梭菌产毒株较之细菌分离培养、细胞毒素测定方法具有快速、简便、特异、敏感的优点，可望用于临床标本的直接检测。     

Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae. Genitourinary infections in males by the Amplicor PCR assay of urine

The Amplicor CT/NG polymerase chain reaction (PCR) test on urine specimens from males was prospectively evaluated against established specimens and laboratory methods for diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae genitourinary infections, in patients from a remote region of Western Australia. Seventy-three males who were tested for both C. trachomatis and N. gonorrhoeae by both conventional methodology and Amplicor PCR on urine were enrolled in the study. Established testing comprised enzyme immunoassay/immunofluorescence antigen testing (EIA/IF) for C. trachomatis and microscopy and/or culture for N. gonorrhoeae on urethral swabs. Positive test results were confirmed using a set of criteria that included supplemental PCR testing and clinical history. Overall, 13.7% of patients were resolved as positive for C. trachomatis and 52.1% as positive for N. gonorrhoeae. The sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens for C. trachomatis were 80.0% (8/10) and 95.2% (60/63), compared with 60.0% (6/10) and 100.0% (63/63) for EIA/IF on urethral swabs. For N. gonorrhoeae, the sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens were both 100% (38/38 and 35/35, respectively) compared with 86.8% (33/38) and 100% (35/35) for microscopy and/or culture on urethral swabs. The results of this study indicate that the Amplicor CT/NG multiplex PCR test for C. trachomatis and N. gonorrhoeae performed on urine in males provides a highly sensitive, specific, and robust method for the diagnosis of both C. trachomatis and N. gonorrhoeae, for the early detection of both symptomatic and asymptomatic infected individuals.     

A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene

Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.     

细胞壁缺陷型淋病奈瑟菌cppB基因酶切图谱分析

目的探讨细胞壁缺陷对淋病奈瑟菌隐蔽性质粒B(cppB)基因的影响以及细胞壁缺陷型淋病奈瑟菌cppB的变异特点。方法用青霉素诱导淋病奈瑟菌成为细胞壁缺陷型并获得其纯培养物,cppB基因特异性引物PCR检测细胞壁缺陷型纯培养物的cppB基因,并对其cppB基因PCR扩增产物进行限制性核酸内切酶图谱分析。结果细菌型和细胞壁缺陷型均具有cppB基因扩增产物,经过限制性内切酶HindⅢ、HinfⅠ、HpaⅡ和MspⅠ消化和电泳,在5%PAGE凝胶电泳中,呈现出相同的电泳条带。结论淋病奈瑟菌细菌型及其细胞壁缺陷型对cppB基因限制性核酸内切酶(HindⅢ、HinfⅠ、MspⅠ、HpaⅡ)的分析没有发现淋病奈瑟菌L型具有与其亲代细菌型不同的图谱,提示细胞壁缺陷没有导致淋病奈瑟菌的cppB基因发生这些核酸酶切位点中核苷酸序列的改变。     

A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler

The detection of Neisseria gonorrhoeae by the polymerase chain reaction (PCR) is now recognized as a sensitive and specific method of diagnosing infection by the organism. In this Study 152 urine specimens were examined for N. gonorrhoeae by a real-time PCR method using the LightCycler platform and results were compared to an "in-house" PCR assay using an ELISA-based detection method. N. gonorrhoeae DNA was detected in 29 (19%) specimens by LightCycler PCR (LC-PCR) and in 31 (20%) specimens by the "in house" PCR method. The LightCycler assay proved to be specific and 94% sensitive when compared to the "in house" PCR method. These features combined with the rapid turn-around time for results makes the LC-PCR particularly suitable for the detection of N. gonorrhoeae in a routine clinical laboratory.     

Evaluation of PorB variable region typing of Neisseria gonorrhoeae using PCR-ELISA in samples collected from men who have sex with men

A polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay (ELISA) methodology was developed to characterize Neisseria gonorrhoeae porB gene variable regions (VR); the methodology was evaluated in comparison to porB VR typing by checkerboard hybridization. Clinical noncultured samples from 35 men who have sex with men (MSM), positive by nucleic amplification assays for N. gonorrhoeae, were typed using a panel of 40 oligonucleotide probes to porB VRs and compared to checkerboard hybridization. Complete concordance was observed between the two methods at PIB VRs 1, 3, and 7. At the more degenerate VRs 5 and 6, PCR ELISA resulted in obtaining more typeable VRs than checkerboard hybridization due to single nucleotide mismatches. By PCR ELISA, two predominant PIB porB types were identified in 58% of the samples and the remaining 16 samples had one of six other porB types. Both PCR ELISA and checkerboard hybridization methods of porB VR typing allowed characterization of N. gonorrhoeae from noncultured clinical samples including throat and rectal swabs and discriminated N. gonorrhoeae from N. meningitidis present in some of the samples. PCR ELISA is a rapid, relatively inexpensive and alternative molecular typing method for N. gonorrhoeae, suitable for use in conjunction with molecular diagnostic tests.     

Rapid detection of Neisseria meningitidis in cerebrospinal fluid by one-step polymerase chain reaction of the nspA gene

A polymerase chain reaction (PCR) protocol for the rapid detection of meningococcal DNA in cerebrospinal fluid (CSF) was developed and optimized. A set of primers based on Neisseria surface protein A (nspA) gene sequence was designed to amplify a 481-bp product specific for N. meningitidis. We tested 85 N. meningitidis strains obtained from patients with meningococcal meningitis and 112 CSF samples from patients with suspected meningococcal meningitis. No amplification of the nspA gene was observed from other Neisseriaceae species (except from N. gonorrhoeae) and from other bacteria frequently associated with meningitis. N. meningitidis belonging to different serogroups yielded the same product after PCR amplification. The sensitivity and specificity of our protocol was determined by comparing the results of specific amplification of nspA gene by PCR reaction (nspA-PCR) with those obtained by conventional methods. All positive samples by conventional methods were confirmed by nspA-PCR, whereas 48% of negative samples after culture and latex agglutination tested positive by nspA-PCR. The use of nspA-PCR proved to be a rapid diagnostic method, in which sensitivity and specificity may not be affected by prior antibiotic treatment.     

Mosaic-like structure of penicillin-binding protein 2 Gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime

Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 micro g/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 micro g/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.     

Relation between genetic markers of drug resistance and susceptibility profile of clinical Neisseria gonorrhoeae strains

The main goal of this work is to clarify the predictive value of known genetic markers of Neisseria gonorrhoeae resistance to penicillin, tetracycline, and fluoroquinolones. The correlation between the presence of certain genetic markers and susceptibility of N. gonorrhoeae isolates to penicillin, tetracycline, and fluoroquinolones has been analyzed by means of statistical methods. Susceptibility testing with penicillin, tetracycline, and fluoroquinolones was performed by the agar dilution method. N. gonorrhoeae genomic DNA was isolated. The presence of bla(TEM-1) and tet(M) genes was analyzed by PCR. A novel method of polymorphism discovery based on a minisequencing reaction followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry was applied for the analysis of chromosomal N. gonorrhoeae genes involved in antimicrobial resistance development. Clinical N. gonorrhoeae isolates (n = 464) were collected. Susceptibility levels to penicillin, tetracycline, and fluoroquinolones were found to be 25.9%, 35.9%, and 54.1%, respectively. Among the 19 N. gonorrhoeae isolates with penicillin MICs of > or =4 microg/ml, the bla(TEM-1) gene was detected in 12. The Tet(M) determinant was found in 4 of 12 N. gonorrhoeae isolates with tetracycline MICs of > or =16 microg/ml. The chromosomal genetic markers of penicillin and tetracycline resistance were detected especially in isolates with penicillin MICs of 0.25 to 2.0 microg/ml and tetracycline MICs of 0.5 to 4 microg/ml. Mutations in GyrA and ParC were found in 208 of 211 quinolone-resistant N. gonorrhoeae isolates. This work is the first representative molecular research of the N. gonorrhoeae population in Russia. Information about the prevalence of antibiotic resistance mechanisms and the positive predictive value of certain genetic determinants is given. The positive predictive values of the analyzed genetic markers were found to be different for fluoroquinolones (90.3%), penicillin (91.1%), and tetracycline (81.9%).     

Oral ciprofloxacin versus ceftriaxone for the treatment of urethritis from resistant Neisseria gonorrhoeae in Zambia.

Neisseria gonorrhoeae strains resistant to treatment with penicillin, tetracycline, and/or spectinomycin are increasing in prevalence in many parts of the world. In Zambia, 52% of N. gonorrhoeae isolates produced beta-lactamase in 1986. Few oral regimens have proven effective for treatment of resistant N. gonorrhoeae. We conducted a prospective, double-blind, randomized clinical trial of 250 mg of ciprofloxacin given orally versus 250 mg of ceftriaxone given intramuscularly for treatment of uncomplicated gonococcal urethritis in adult males. Two hundred men were enrolled and treated. The two groups were comparable in age (27.5 years), prevalence of latent syphilis (14 and 10%), and human immunodeficiency virus infection (32 and 38%). Of 165 patients with cultures positive for N. gonorrhoeae who returned for follow-up, ciprofloxacin cured 83 of 83 (100%), including 26 with penicillinase-producing N. gonorrhoeae (PPNG) and 21 with N. gonorrhoeae with chromosomally mediated resistance to multiple antibiotics (CMRNG), and ceftriaxone cured 81 of 82 (98.7%), including 30 with PPNG and 19 with CMRNG. Both treatment regimens were well tolerated. Chlamydia trachomatis in urethral exudate was found by direct fluorescent-antibody microscopic examination or by culture in 10 (5%) participants. All N. gonorrhoeae isolates were inhibited by ceftriaxone at 0.06 micrograms/ml, except one which was inhibited at 0.125 micrograms/ml, while ciprofloxacin inhibited all isolates at 0.03 micrograms/ml. Ciprofloxacin is a safe and effective therapy for uncomplicated gonococcal urethritis, including that caused by PPNG and CMRNG in human immunodeficiency virus-infected men.     

A real-time PCR assay for the detection of Neisseria gonorrhoeae in genital and extragenital specimens

A Neisseria gonorrhoeae LightCycler (NGpapLC) assay targeting the porA pseudogene was compared with bacterial culture for detection of N. gonorrhoeae in 636 clinical specimens (216 cervical, 185 urethral, 196 throat, and 39 rectal swab specimens). The specificity of the NGpapLC assay was further investigated by testing a bacterial reference panel comprising several Neisseria species. Overall, 19 (3.0%) specimens were positive and 613 (96.4%) specimens were negative by both methods. Four (0.6%) specimens were positive by the NGpapLC assay only. For the cervical and urethral swabs, the NGpapLC provided 100% sensitivity and 100% specificity compared with bacterial culture. Following discrepant analysis, the clinical sensitivity and specificity of the NGpapLC for throat and rectal swabs was also 100%. For the bacterial panel, only N. gonorrhoeae isolates provided positive results. The results show the NGpapLC assay is suitable for use on a range of clinical specimens and could improve detection of pharyngeal N. gonorrhoeae.