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。     

OmpA-VS2为靶基因的非标记实时荧光PCR检测沙眼衣原体感染

目的建立一种以OmpA-VS2为靶基因的非标记实时荧光聚合酶链反应(PCR)方法,以检测沙眼衣原体。方法设计15种型别沙眼衣原体通用引物,对OmpA-VS2进行套式PCR扩增和实时荧光PCR检测,并对其敏感性、特异性进行评价,并用临床标本进行验证。结果15种型别沙眼衣原体均扩增出168bp的目的片段;敏感性为每个反应1个拷贝。特异性分析可见,与10种泌尿生殖道病原体和共生菌均没有交叉反应。临床标本验证的结果显示,该方法与以质粒为靶基因的核酸扩增检测的Amplicor CT/NG方法的检测结果一致。结论该方法具有敏感性高、特异性强、扩增后无需PCR后处理等特点,可望用于临床与防治项目中沙眼衣原体的检测。     

聚合酶链反应检测淋病患者生殖道合并感染沙眼衣原体

淋病是我国目前常见的性传播疾病，淋病患者合并沙眼衣原体泌尿生殖道感染在临床上也很普遍。据国外有关文献报道，美国淋病患者合并沙眼衣原体泌尿生殖道感染的发病率为12％～15％'[1]。国内在这方面的流行病学资料甚少，其主要原因是缺乏简便、可靠的衣原体实验室检测方法以及单纯从临床症状和体征上对淋病和衣原体感染进行区别具有一定的困难。近几年发展起来的聚合酶链反应（PCR）具有特异性强、敏感性高的优点，国外已相继建立了诊断沙眼衣原体的各种方法。我们用自己设计的引物建立的PCR方法对我院门诊确诊为淋病（细菌培养法）的…     

PCR技术在男性泌尿系感染的临床应用

淋球菌（NG）、解脲支原体（UU）和沙眼衣原体（CT）是引起男性泌尿生殖系感染最常见的病原体。这些感染在临床上非常普遍。以往的检测方法有细胞培养、免疫荧光、酶联免疫等，这些方法繁琐、费时、阳性率低、灵敏度和特异性也低．而PCR技术与传统方法的比较．有简便、快速、敏感、持异等优点，提高了临床病原微生物实验诊断的准确性及灵敏度。为了协助临床诊断和观察治疗结果，我们将PCR技术用于男性泌尿系炎症的淋球菌、解脲支原体、沙眼衣原体372例的检测，现报告如下。一、材料与方法（一）仪器设备：扩增仪为美国PE公司TCI循环…     

衣原体感染

沙眼衣原体的快速诊断试验(译文)——栗克喜等(四川 成都生物制品研究所)；《国外医学&;#183;微生物学分册》，2003，26(6)：30[沙眼衣原体是性传播性疾病的重要病原体，本文阐述其多种筛选的快速诊断方法。高敏感性和特异性的分子生物学方法有助于临床的快速诊断，这些方法     

衣原体感染

HPV16、沙眼衣原体、生殖道支原体感染与子宫颈癌关系的研究；沙眼衣原体K血清型感染细胞IL-6分泌水平的测定；荧光定量PCR和超高倍显微诊断系统检测沙眼衣原体；口服药物联合奥平栓治疗生殖道衣原体、支原体感染的疗效分析[编按]     

沙眼衣原体细胞培养法在临床诊断中的应用

泌尿生殖道沙眼衣原体感染是近年来在我国发病率迅速上升的性传播疾病之一。它引起的疾病范围广泛，并能导致诸如盆腔炎及异位妊娠等严重并发症。因此，如何正确诊断该病就显得十分必要。实验室诊断方法能够发现有症状或无症状感染者的病原体，国内已开展的有聚合酶链反应（PCR）法、直接免疫荧光法（DFA）、酶免疫法（EIA）和细胞培养等。我们开展沙眼衣原体细胞培养已有十几年，[第一段]     

衣原体感染

肺炎衣原体感染致C57BL／6J小鼠内皮功能异常；肺炎衣原体感染和高脂血症对心肌细胞NF-kappa B和AP-1的影响；956例阴道分泌物标本沙眼衣原体检测结果分析；沙眼衣原体感染输血传播病毒感染状况调查；三种沙眼衣原体检测方法的对比评价.     

酶联免疫吸附试验和聚合酶链反应检测生殖器标本中的单纯疱疹病毒

目的 评价酶联免疫吸附试验(ELISA)诊断生殖器疱疹的临床应用价值。方法 对120例具有生殖器皮肤、粘膜损害的病人，采用ELISA和分型聚合酶链反应(PCR)检测其生殖器标本中的单纯疱疹病毒(HSV)，两种试验结果不符合者采用第二种PCR检测。结果 120例受检者中，ELISA检出49例HSV阳性，分型PCR检出50例阳性。单纯HSV-1感染者占生殖器疱疹的10.0％，单纯HSV-2感染者占80.0％，HSV-1和HSV-2混合感染者占10.0％。在120例标本中，107例两种试验结果是符合的，13例结果不符合。与“扩大金标准”比较，ELISA的敏感性、特异性、阳性预期值分别为92.0％、95.7％、93.8％，分型PCR的敏感性、特异性、阳性预期值分别为94.0％、95.7％、94.0％。结论 ELISA法检测HSV感染，其敏感性和特异性高，方便、快速，尤其适合大批量样本的检测，值得在临床应用。     

聚合酶链反应诊断沙眼衣原体感染

聚合酶链反应诊断沙眼衣原体感染王宁遂，朱静，邓兵，梁永霓作者采用聚合酶链反应（ＰＣＲ）扩增沙眼衣原体ＰＣＴＴ１质粒特异性ＤＮＡ片段，并用双链ＤＮＡ循环测序鉴定扩增产物，使病原检测的特异性明显提高，对衣原体的早期诊断有重要意义，现总结报道如下。材料和方...     

Polymerase chain reaction diagnosis of Helicobacter pylori in gastroduodenal diseases: Comparison with culture and histopathological examinations

Helicobacter pylori has been associated with a variety of upper gastrointestinal diseases. Histopathological examination and culture are considered to be the more specific tests in the diagnosis of H. pylori infection. In the present study, we evaluated the efficiency of a polymerase chain reaction (PCR) assay of the H. pylori urease A gene as a procedure in the diagnosis of gastric H. pylori infection in various gastroduodenal diseases. Biopsy specimens were obtained from the antral mucosa of 83 patients during endoscopic examination and were submitted to three tests for the detection of H. pylori infection. The detection rates of H. pylori using PCR, histopathological examination and culture were 84, 77 and 63%, respectively. When the infection was defined, by the agreement of culture and histopathological examination or by positive culture, the PCR assay had a sensitivity of 98.1% and a specificity of 84.6%. When the infection was defined by a positive result of either two of the three tests or by positive culture, the PCR assay had a sensitivity of 98.6% and a specificity of 85.7%. We conclude that the PCR assay is a valuable test for the diagnosis of H. pylori infection in gastroduodenal diseases.     

Screening for Chlamydia trachomatis infection in college women with a polymerase chain reaction assay.

This study sought to determine factors associated with chlamydial infection in a low-prevalence college health setting and to determine the testing characteristics of a polymerase chain reaction (PCR) assay for chlamydial infection (AMPLICOR chlamydia test; Roche Diagnostic Systems, Indianapolis) in this population. Young women (n = 1,149) at a university student health clinic underwent testing for cervical chlamydial infection by PCR assay and culture; the characteristics of women with and without chlamydial infection were compared. Chlamydial infection was diagnosed for 26 students (2.3%). The sensitivity and specificity of PCR assay and culture were 85% and 100% and 54% and 100%, respectively. Students with chlamydial infection were more likely to be 20 years of age or younger, have symptoms, report prior chlamydial infection or gonorrhea, report exposure to a sexually transmitted disease (STD), be black, or have cervical signs during examination; however, none of these were significant predictors for asymptomatic women. PCR assay detected significantly more cervical infections than did culture in this college student population. These data are consistent with recommendations for testing college women with symptoms, STD exposure, or age of younger than 25 years.     

Rapid diagnosis of cytomegalovirus pneumonia in marrow transplant recipients by bronchoalveolar lavage using the polymerase chain reaction, virus culture, and the direct immunostaining of alveolar cells

The objective was to compare the use of the polymerase chain reaction (PCR), virus culture, and immunostaining of alveolar cells used alone and in combination as diagnostic methods for the rapid diagnosis of cytomegalovirus (CMV) pneumonia in marrow transplant recipients. Seventy-five marrow transplant recipients with clinical and radiological evidence of pneumonitis were used as subjects. Bronchoalveolar lavage was performed on all patients to obtain material for conventional and/or rapid CMV culture, immunostaining of alveolar cells with monoclonal antibodies (MoAbs), and amplification of CMV-DNA by PCR. Assay results were then prospectively correlated with clinical outcome. Seven of the 75 patients (9.3%) had CMV pneumonitis and 6 patients (8%) had CMV infection without pneumonia. PCR is the most sensitive assay for the detection of CMV in bronchoalveolar lavage fluid. For the diagnosis of CMV pneumonitis, the sensitivity of alveolar cell immunostaining and PCR were both 100%. The sensitivity of virus culture was 85.7%. The positive predictive value for each test, used alone, for the identification of CMV pneumonitis was low. However, when the result of the PCR assay was assessed in combination with CMV immunostaining of alveolar cells, the sensitivity, specificity, positive, and negative predictive value of this strategy was 100%. The concomitant use of PCR and the rapid immunostaining of alveolar cells for CMV has facilitated the development of a sensitive and specific diagnostic algorithm for the detection and early treatment of CMV pneumonitis in transplant recipients.     

The Cobas Amplicor MTB test for detection of Mycobacterium tuberculosis complex from respiratory and non-respiratory clinical specimens

The Cobas Amplicor MTB test is a polymerase chain reaction (PCR) technique commonly used for direct detection of Mycobacterium tuberculosis in clinical samples. This assay is only validated for respiratory specimens, but many physicians also request PCR analyses for non-respiratory ones. 877 respiratory and 564 non-respiratory specimens were analysed by this test. Using culture results as standard, the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of the PCR were, respectively, 97.9%, 100%, 100% and 94.4% for smear-positive respiratory specimens, 68.8%, 99.2%, 87.5% and 97.5% for smear-negative respiratory samples, 57.8%, 98.6%, 78.8% and 96.4% for all non-respiratory specimens, and 42.4%, 98.6%, 66.7% and 96.4% for smear-negative non-respiratory specimens. 154 cerebrospinal fluid samples were analysed and the sensitivity, specificity, PPV and NPV were 55.6%, 97.2%, 55.6% and 97.2%, respectively. These results indicate that the Cobas Amplicor MTB test enables detection of tuberculosis in respiratory specimens, but does not perform well enough in non-respiratory specimens. The method fails particularly in cases where a reliable and rapid test is urgently needed, e.g. in tuberculous meningitis.     

Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women

Group B Streptococcus is a causative agent of invasive neonatal infections. Maternal colonization by Streptococcus agalactiae is a necessary condition for vertical transmission, with efficient screening of pregnant women playing an essential role in the prevention of neonatal infections. In this study, we aimed to compare the performance of conventional polymerase chain reaction and real-time PCR assays as screening methods for S. agalactiae in pregnant women against the microbiological culture method considered as the gold-standard. A total of 130 samples from pregnant women were analyzed for sensitivity, specificity, positive predictive value, and negative predictive value. Statistical analysis was performed using the SPSS software, version 20.0. The verified colonization rate was 3.8% with the gold-standard, 17.7% with conventional PCR assay, and 29.2% with the real-time PCR test. The trials with conventional PCR and real-time PCR had a sensitivity of 100% and a specificity of 85.6% and 73.6%, respectively. The real-time PCR assay had a better performance compared to the gold-standard and a greater detection rate of colonization by S. agalactiae compared to conventional PCR assay. With its quick results, it would be suitable for using in routine screenings, contributing to the optimization of preventive approaches to neonatal S. agalactiae infection.     

Efficacy of Amplicor PCR for the diagnosis of tuberculosis in respiratory specimens other than sputum.

A total of 832 respiratory specimens not including the sputum (402 bronchial lavages, 241 bronchial brushing specimens, 136 pumping lavages, 41 pleural effusions, and 12 others) from 462 patients were assayed using the Roche Amplicor Mycobacterium tuberculosis test for amplification and identification of M. tuberculosis, M. avium and M. intracellulare (Amplicor PCR). The results were compared with those obtained using conventional microscopy and cultivation methods. Each patient had little or no sputum and showed an abnormal chest X-ray shadowing of unknown cause. No patients had previously undergone antituberculous therapy. Of the specimens obtained, 24 were both PCR and culture positive, 786 were both PCR and culture negative, 11 were PCR positive and culture negative, and 11 were PCR negative and culture positive. Based on these results, the sensitivity and specificity of Amplicor PCR were determined to be 68.67% and 98.6%, respectively, when compared with culture of respiratory specimens not including the sputum. After correcting for discrepancies due to differences in patient clinical data, the sensitivity of Amplicor PCR was found to be 68.6%, and the specificity to be 99.9%; the corresponding values for culture were 66.7% and 100%, and those for smear were 9.8% and 100%. Thus, Amplicor PCR was shown to possess a similar sensitivity to culture and to be a highly specific technique for the diagnosis of tuberculosis in the respiratory system using non-sputum specimens within hours in patients showing little or no sputum and abnormal chest X-ray shadowing of an indeterminant cause.     

Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva

Background

Low levels of Cytomegalovirus (CMV) viral load are frequently detected following allogeneic stem cell transplantation (SCT) and CMV disease may still develop in some allogeneic SCT patients who have negative pp65-antigenemia (pp65-Ag) or undetectable DNA. Pp65Ag is a sensitive method to diagnose CMV infection. Quantitative CMV-DNA PCR assay in plasma has been proposed to monitor CMV infection in SCT patients. We evaluated the clinical utility of pp65Ag and PCR assay in plasma of SCT recipients.

Methods

In a prospective longitudinal study, 38 consecutive patients at risk of CMV infection (donor and/or recipient CMV seropositive) were weekly monitored for CMV infection by both quantitative CMV-PCR in plasma (COBAS AMPLICOR CMV MONITOR) and pp65 Ag, during the first 100 days after SCT.

Results

A total of 534 blood samples were simultaneously analysed for pp65Ag and PCR. Overall, 28/38 patients (74%) had active CMV infection within 100 days from SCT. In 16 patients, CMV was first detected by pp65 Ag alone; in 5 patients by both methods and in 6 by PCR assay alone; one patient had CMV biopsy-proven intestinal disease without pp65Ag and PCR assays positivity before CMV disease. Overall, three patients developed intestinal CMV disease (7.9%): one had negative both pp65Ag and PCR assays before CMV disease, one had disease and concomitant positivity of both methods, while in the remaining patient, only pp65Ag was positive before CMV disease.

Conclusion

Plasma PCR(COBAS AMPLICOR CMV MONITOR) and pp65Ag assays were effective in detecting CMV infection, however, discordance between both methods were frequently observed. Plasma PCR and pp65Ag assays may be complementary for diagnosis and management of CMV infection.     

Reliability of laboratory markers of HIV-1 infection in Argentinian infants at risk of perinatal infection

Early and accurate diagnosis of HIV-1 infection in infants born to HIV-1-seropositive mothers is of great importance. Polymerase chain reaction (PCR), HIV culture, and p24 antigen detection assays were evaluated for their ability to detect the presence of HIV in 195 infants at risk of perinatal infection. Using the Centers for Disease Control and Prevention guidelines for assessing HIV infection status in children younger than 18 months, 70 infants (36%) were diagnosed as HIV-1 infected and 125 (64%) lacked virologic and clinical evidence of infection. PCR and HIV culture were the most sensitive laboratory markers, detecting 100% and 98% of positive samples, respectively, regardless of age at testing. HIV-1 p24 antigen assay was detected in 26 of 38 positive samples but not in negative samples. PCR was performed with three different sets of primers (SK38/SK39-SK19-gag, SK68/SK69-SK70-env, and SK150/SK431-SK102-gag). The sensitivity/specificity of the individual assays were for SK19, 96.1%/94.25%; SK70, 89.6%/100%; and SK102, 100%/100%. A sample was considered HIV-1 positive when two positive PCR results were obtained with two different pairs of primers, and negative if the sample was negative when three sets of primers were used. False-positive results were occasionally obtained with probe SK19 in six seroreverter infants before serologic status was known. This suggested that the infection was caused by nonreplicative strains or were false-positive results probably by nonspecific amplification due to cross-reaction with other microorganisms; contamination was discarded because there was no specific amplification with the other two primers. All the HIV-1-infected infants were correctly identified with PCR; all except one could be identified with coculture and only 68.4% were confirmed with p24 antigen assay. No seroreverter infant was misdiagnosed using the criteria selected.     

聚合酶链反应技术与镜检法诊断间日疟的对比研究

目的　评价聚合酶链反应技术在间日疟诊断中的应用价值。　方法　根据红内期疟原虫 SSUr RNA编码基因序列 ,设计合成 1对引物 ,采用聚合酶链反应技术 ,扩增检测“四热”病人血样中间日疟原虫 DNA;同时作厚血膜镜检疟原虫。　结果　从间日疟患者血样中扩增出 341bp的 DNA片段 ,与预期的扩增片段大小相符 ,而正常人血样及空白对照无特异性条带 ;对于 2 34份“四热”病人血样 ,PCR法检出 16份阳性 ,阳性率为 6 .8% ;厚血膜镜检 13份阳性 ,阳性率为 5 .6 % ;两种方法相比差异无显著性 (P>0 .0 5 ) ;对于 3份 PCR阳性而镜检法阴性的血样 ,重新作 PCR,结果仍然为阳性。以镜检法为标准 ,PCR法的灵敏度为 10 0 % ,特异度为 98.6 %。　结论　PCR技术灵敏特异 ,可替代镜检法用于间日疟感染的临床及现场检测。     

A newly developed PCR assay ofH. pylori in gastric biopsy,saliva, and feces

We have recently developed a new PCR assay for the detection ofH. pylori. In this study, the polymerase chain reaction (PCR) assay was used to detectH. pylori in 88 gastric biopsy, 85 saliva, and 71 fecal specimens from 88 patients.H. pylori infection was confirmed in 71 of 88 patients by culture and/or histological stain of gastric biopsies. Serum IgG antibody toH. pylori was also measured and resulted in 97% sensitivity and 94% specificity.H. pylori DNA was detected by the PCR assay in gastric biopsy specimens from all 71 patients (100% sensitivity) with proven gastricH. pylori infection but not from 17 noninfected patients (100% specificity). In saliva specimens,H. pylori DNA was identified in 57 of the 68 patients (84%) with proven gastricH. pylori infection and in three of the 17 patients without gastricH. pylori infection. However, the PCR assay was only able to detectH. pylori DNA in the feces from 15 of 61 patients (25%) with proven gastricH. pylori infection and one of the 10 patients without gastricH. pylori infection. The results show that the PCR assay is reliable for detecting the presence ofH. pylori in gastric biopsy and saliva specimens. The data indicate thatH. pylori exists in a higher prevalence in saliva than feces and that the fecal-oral route may be an important means of transmission of this infection in developing countries but not as significant as previously suspected in the developed countries. It is likely that the oral-oral route is more prominent.