实时荧光 PCR 技术和细菌培养法检测妊娠晚期孕妇定植B 群链球菌临床分析

目的：探讨应用实时荧光 PCR 技术和细菌培养法检测妊娠晚期孕妇定植 B 群链球菌（GBS）的敏感性。方法采集孕妇生殖道-直肠分泌物拭子2份,一份标本用普通细菌培养法、另一份用实时荧光 PCR 技术检测生殖道 GBS。比较两种方法的准确性、快速性。308例孕妇根据实时荧光 PCR 检测分为 GBS 阳性组与 GBS 阴性组,通过对比较分析 GBS 与发生胎膜早破的关系。结果将308例孕妇进行生殖道 GBS 检测,用普通细菌培养法进行培养有18例阳性,阳性率5．8％（18/308）,实时荧光 PCR检测有29例阳性,阳性率9．4％（29/308）。在 PCR 检测 GBS 阳性组中,发生胎膜早破9例,发生率为31％。在 PCR 检测 GBS阴性组中,发生胎膜早破33例,发生率为11．83％。结论该次调查结果显示实时荧光 PCR 技术的阳性检出率明显高于细菌培养法,应用该法检测技术为 GBS 的快速诊断以及更加准确有效地进行抗菌药物预防提供了依据。     

孕妇生殖道B群链球菌快速检测方法的建立和初步应用

目的建立一种快速、灵敏、特异的PCR检测孕妇生殖道GBS感染的方法。方法根据GBS保守序列:cfb设计特异性扩增引物,优化PCR反应,通过电泳判断结果,同时使用UNG酶,dUTP避免PCR产物污染。结果本研究通过PCR条件的反复摸索,优化了PCR检测方法。同微生物培养方法相比,PCR方法灵敏度更高,检测更快速。直接煮沸法和试剂盒法的DNA提取效率具有差别,试剂盒法更适合临床诊断使用。结论本研究成功建立了快速、灵敏、特异的孕妇生殖道GBSPCR检测方法。     

Comparison of BD GeneOhm real-time polymerase chain reaction with chromogenic and conventional culture methods for detection of group B Streptococcus in clinical samples

A total of 200 antenatal high vaginal swabs were screened for the presence of group B Streptococcus (GBS) using a conventional culture method (recommended by Centers for Disease Control and Prevention). Screening was also performed by using a new chromogenic agar, chromID Strepto B, and by using the BD GeneOhm StrepB real-time polymerase chain reaction (PCR), which was performed directly on swabs without enrichment. Using a combination of all methods, we detected GBS in 101 samples. A total of 82 samples (81.2%) were positive using PCR, and 83 samples (82.2%) were confirmed as positive by culture (any method). PCR was more sensitive for detection of GBS than direct culture using any method (P < 0.0005). PCR was also more sensitive than any single enrichment method, but this difference was not statistically significant. With culture as a "gold standard", the PCR method showed a sensitivity of 77.1% and a positive predictive value of 79.3%. Of the culture-positive samples, significantly, more GBSs were detected by direct plating on chromID Strepto B than on selective sheep blood agar (67.5% versus 57% respectively, P < 0.02). After selective enrichment, 92.8% of GBS were isolated on chromID Strepto B compared with 89.2% isolated on sheep blood agar.     

Evaluation of a real-time fluorescent PCR assay for rapid detection of Group B Streptococci in neonatal blood

Streptococcus agalactiae (Group B Streptococcus: GBS) is the major causative agent of neonatal sepsis. Neonates at risk for GBS infections are empirically administered broad-spectrum antibiotics for at least 48 h pending blood culture results. A rapid assay to expedite detection of GBS would facilitate initiation of specific antibiotic therapy. Conversely, expeditious proof of absence of infection will avoid unnecessary antibiotic use. Using the LightCycler, we evaluated a hybridization probe polymerase chain reaction (PCR) assay to detect GBS-specific cfb gene target DNA sequence in blood specimens. Both sensitivity and specificity of the real-time PCR assay was 100%. The assay demonstrated 100% specificity when tested against 26 non-GBS bacteria. This method is capable of detecting as few as approximately 100 copies or 10 pg of GBS genomic DNA. This real-time PCR method is rapid, sensitive, and specific for the detection of GBS in neonatal blood samples and holds great promise in its utility in the diagnostic laboratory.     

建立实时荧光LAMPA法检测肺炎支原体

目的:建立实时荧光环介导等温扩增(LAMP)法检测肺炎支原体。方法:在LAMP体系中添加适量荧光染料SYTO-9,通过实时定量PCR仪监测LAMP反应结果。以肺炎支原体P1基因重组质粒制备一系列浓度标准品,分别使用实时荧光LAMP法和SYBR GreenⅠ染料LAMP法检测,比较2种方法的检出限。肺炎支原体M129标准株菌液以10倍梯度稀释,分别以实时荧光LAMP法和肺炎支原体荧光PCR试剂盒检测,比较2种方法灵敏度。以实时荧光LAMP法检测常见呼吸道感染病原体肺炎链球菌、金黄色葡萄球菌、表皮葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和大肠埃希菌基因组DNA,评估该方法特异性。采集疑似肺炎支原体感染患儿咽拭子标本,分别以实时荧光LAMP法和荧光PCR法检测,比较2种方法检出率差异。结果:实时荧光LAMP法可以检出P1基因最低限为103 copies/μL,当基因拷贝数大于104 copies/μL时,反应30 min样本组即可出现明显荧光信号。SYBR GreenⅠ染料LAMP法可以检出P1基因最低限为104 copies/μL,反应需1 h才可获得足够产物用于显色。实时荧光LAMP法灵敏度高于实时定量PCR法100倍,且对常见呼吸道病原体DNA无扩增。实时荧光LAMP法和荧光PCR法检测疑似肺炎支原体感染咽拭子标本的阳性率差异无统计学意义(P>0.05)。结论:实时荧光LAMP法可以有效避免因开盖检测造成的气溶胶污染,反应时间短,灵敏度高,适于在基层医院中推广使用。     

HBV-HCV-HIV三联荧光PCR检测的内标浓度选择研究

目的选择一个在乙型肝炎病毒-丙型肝炎病毒-人类免疫缺陷病毒(HBV-HCV-HIV)三联荧光PCR检测试验中既可有效监控假阴性结果出现,又对阳性结果影响最小的内标浓度。方法应用不同浓度的内标参入酶链聚合反应(PCR)反应,确定最佳内标参入量;并在适量内标浓度下,检测低浓度标本的阳性检出率。结果由内标浓度5拷贝/PCR、10拷贝/PCR、20拷贝/PCR、50拷贝/PCR、100拷贝/PCR 5个浓度中,优选出最适的内标浓度为20拷贝/PCR,此条件下,阴性标本的内标检出率为100%,检测灵敏度标本的阳性检出率与无内标样本差异无统计学意义。结论合适浓度的内标参与荧光PCR检测能有效地解决了每个标本的质控问题,指示反应体系(试剂耗材)与检测体系(仪器)的有效性。     

GeneXpert实时荧光定量PCR快速检测艰难梭菌的临床评估

目的对GeneXpert实时荧光定量聚合酶链反应(PCR)在快速检测临床粪便标本中艰难梭菌的应用进行评估。方法采用双拭子蘸取临床未成形粪便标本,一支拭子用于GeneXpert实时荧光定量PCR检测艰难梭菌毒素基因tcdB,另一支用于常规厌氧菌培养检测;对GeneXpert实时荧光定量PCR检测结果与常规厌氧菌培养结果的一致性进行统计学分析,并计算GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值等参数。结果临床收集到141例未成形粪便标本,GeneXpert实时荧光定量PCR检出艰难梭菌毒素基因tcdB阳性42例,其中常规厌氧菌培养阳性34例,两者一致性较好(Kappa=0.775 0,P0.01),GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为87.2%、92.2%、81.0%和94.9%。结论 GeneXpert实时荧光定量PCR直接检测粪便标本中的艰难梭菌具有检测快速、操作简便等优点,有重要的临床应用价值。     

实时荧光PCR法和流式细胞术检测HLA-B27的结果分析

目的比较实时荧光PCR法和流式细胞术两种方法检测人类白细胞抗原B27(HLA-B27)的临床应用价值。方法分别采用实时荧光PCR法和流式细胞术两种方法检测225例疑似强直性脊柱炎患者血中HLA-B27,比较和分析两种方法的检测结果。结果 95.11%样本检测结果相同,差异不具有统计学意义(P0.05),将存在差异的标本进行基因测序后,结果与实时荧光PCR的检测结果一致。结论两种方法检测HLA-B27均具有较高的灵敏度和特异度,实时荧光PCR法在结果的准确性上更优于流式细胞术。     

海洋创伤弧菌LAMP快速诊断方法的建立与评价

目的利用环介导等温扩增(LAMP)技术,建立海洋创伤弧菌快速、简便、特异且敏感的检测方法,并对该方法的特异性和灵敏度进行评价。方法选取海洋创伤弧菌溶细胞素(vvhA)基因作为靶基因,根据GenBank公布的序列设计4条LAMP引物;对47株细菌(包括20株弧菌属细菌)进行LAMP和聚合酶链式反应(PCR)扩增,并做特异性比较;对创伤弧菌M06株增菌液10倍倍比稀释,提取DNA后进行灵敏度的检测,并与常规PCR作比较;构建含vvhA基因片段的重组质粒,作为LAMP反应体系的标准阳性对照。结果常规PCR实验出现假阳性结果,而LAMP实验只有海洋创伤弧菌出现阳性扩增,其他样品均为阴性,无假阳性和假阴性结果,表明引物的特异性较好,加入钙黄绿素和电泳结果相一致;针对vvhA基因建立的LAMP技术其最低检测下限为每个反应4×10CFU,是常规PCR(每个反应4×10~2 CFU)的10倍,呈现较好的灵敏度。重复实验过程两遍,PCR和LAMP技术检测结果稳定。结论建立了一种用于检测海洋创伤弧菌的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,特别适合用于现场和床旁的快速检测。     

Sensitively and quickly detecting Vibrio vulnificus by real time recombinase polymerase amplification targeted to vvhA gene

Vibrio vulnificus (V. vulnificus) is a Gram-negative bacterium living in warm and salty water. This marine bacterium could produce hemolysin (VVH), which often causes serious gastroenteritis or septicemia when people contact to seawater or seafood containing V. vulnificus. Timely diagnosis is regard as essential to disease surveillance. In this paper, we aimed at developing a quick and sensitive method for the detection of Vibrio vulnificus using real time recombinase polymerase amplification (real time RPA). Specific primers and an exo probe were designed on the basis of the vvhA gene sequence available in GenBank. Target DNA could be amplified and labeled with specific fluorophore within 20 min at 38 °C. The method exhibited a high specificity, only detecting Vibrio vulnificus and not showing cross-reaction with other bacteria. The sensitivity of this method was 2 pg per reaction (20 μL) for DNA, or 200 copies per reaction (20 μL) for standard plasmid. The detection limit (LOD) stated as the target level that would be detected 95% of the time and estimated was 1.58 × 10² copies by fit of the probit to the results of 8 replicates in different concentration. For quantitative analysis of the real time RPA, the second order polynomial regression was adopted in our study. The results showed the correlation coefficients were raised above 0.98, which suggested this model might be a better choice for the quantitative analysis of real time RPA compared to the routine linear regression model. For artificially contaminated plasma samples, Vibrio vulnificus could be detected within 16 min by real time RPA at concentration as low as 1.2 × 10² CFU/mL or 2.4 CFU per reaction (20 μL). Thus, the real time RPA method established in this study shows great potential for detecting Vibrio vulnificus in the research laboratory and disease diagnosis.     

Development and comparative evaluation of real-time PCR and real-time RPA assays for detection of tilapia lake virus

Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Early and accurate diagnosis is an important priority for TiLV disease control. In order to evaluate the methodology in the molecular diagnosis of TiLV, we compared newly developed quantitative real-time PCR (qPCR) and real-time recombinase polymerase amplification (real-time RPA) assays regarding their sensitivities, specificities and detection effect on clinical samples. Real-time RPA amplified the target pathogen in less than 30 min at 39 °C with a detection limit of 620 copies, while qPCR required about 60 min with a detection limit of 62 copies. Both assays were specific for TiLV and there were no cross-reactions observed with other common fish pathogens. The assays were validated using 35 tissue samples from clinically infected and 60 from artificially infected animals. The sensitivities for the real-time RPA and qPCR assays were 93.33 and 100%, respectively, and the specificity was 100% for both. Both methods have their advantages and can play their roles in different situations. The qPCR is more suitable for quantitative analysis and accurate detection of TiLV in a diagnostic laboratory, whereas real-time RPA is more suitable for the diagnosis of clinical diseases and preliminary screening for TiLV infection in poorly equipped laboratories as well as in fish farms.     

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.     

双重TaqMan实时荧光嵌套PCR快速检测α地中海贫血SEA缺失方法的建立与应用

目的 建立一种快速检测α地中海贫血(α地贫)SEA缺失的双重TaqMan实时荧光嵌套PCR方法.方法 收集2010年5-7月在广州市天河区妇幼保健院进行地贫筛查的外周血标本100份,2010年12月至2011年2月东莞东华医院进行产前诊断的胎儿标本7份(绒毛2份、羊水5份).从本实验室样本资源库中选取α地贫SEA缺失已知基因型标本50份.采用双重TaqMan实时荧光嵌套PCR技术,于同一检测体系同时检测各标本d地贫SEA缺失截短序列及缺失范围内正常序列,根据荧光PCR阳性扩增结果结合其Ct值差异诊断受检个体的α地贫SEA缺失基因型.同时采用检测α地贫SEA缺失的常规gap-PCR法,以PCR扩增结合产物凝胶电泳分析各标本α地贫SEA缺失基因型,以验证及对比分析新方法的准确性与实用性.结果 所建立的双重TaqMan实时荧光嵌套PCR优化体系中2个PCR的扩增效率分析曲线的斜率分别为-3.153(SEA缺失截短目的 片段)和-3.182(正常内参序列),扩增效率均接近100%.应用此方法检测50份α地贫SEA缺失已知基因型标本,结果显示该方法不但能实现其快速分子诊断,而且能准确判断受检标本中的外源性污染,从而有效避免假阴性或假阳性误诊.100份外周血标本中,两种方法分别检出SEA缺失标本11份,对比分析两方法的诊断符合率为100%.7份胎儿标本中,gap-PCR法检出SEA缺失3份,而本方法则检出SEA缺失标本2份、受-SEA/αα母源性污染的基因型为αα/αα的绒毛标本1份.结论 本研究建立的双重TaqMan实时荧光嵌套PCR可以快速准确检测α地贫SEA缺失,操作简单实用,适合大规模人群筛查和常规分子诊断.     

Comparison of culture and 2 real-time polymerase chain reaction assays to detect group B Streptococcus during antepartum screening

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates but is preventable if the mother is diagnosed before and treated at delivery. Using 200 vaginal-rectal swabs inoculated to enrichment (LIM) broths, we compared routine culture and 2 real-time polymerase chain reaction (PCR) assays for detection of GBS: the LightCycler (LC) Strep B analyte-specific reagents (ASRs) (Roche Diagnostics, Indianapolis, IN) and the BD GeneOhm StrepB (BD-StrepB) test (BD GeneOhm Sciences, San Diego, CA). Culture detected 26.5% GBS-positive specimens, whereas the LC Strep B ASR and BD-StrepB test identified 29.5% and 30.0% positive specimens, respectively. Because of the increased detection rate of 3.0% to 3.5% observed with PCR, a second GBS-specific amplicon was sequenced to confirm the presence of GBS that was not detected by culture. In our hands, the sensitivity/specificity of the LC Strep B ASR was 100%/95.9%, and the BD-StrepB test was 92.5%/92.5% using culture as the gold standard.     

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 10² copies/μL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.     

布鲁菌PCR快速检测方法的建立及应用

目的建立布鲁菌聚合酶链反应(PCR)方法,对其进行应用评价。方法针对布鲁菌外膜蛋白编码基因(omp-2)设计PCR引物,建立PCR方法,利用19种常见细菌和布鲁菌株DNA分别对其进行特异性和敏感度评价,对3株疑似布鲁菌株进行核酸检测,并与分离培养法进行结果比对。结果本研究建立的布鲁菌PCR检测方法仅能检出布鲁菌阳性菌株,对照菌DNA未出现目的条带;敏感度为100拷贝/反应;PCR方法对3株疑似布鲁菌株检出omp-2基因条带,鉴定结果为布鲁菌,与分离培养法结果相同。结论本研究建立了一种布鲁菌PCR检测方法,与分离培养法相比,布鲁菌PCR方法准确、快速,适合布鲁菌病疫情的快速检测。     

Evaluation of the Xpert® group B streptococcus real-time polymerase chain reaction assay compared to StrepB Carrot Broth™ for the rapid intrapartum detection of group B streptococcus colonization

Xpert group B streptococcus (GBS) was compared to StrepB Carrot Broth™ (SCB) for the detection of intrapartum GBS colonization by dually collecting vaginal/rectal swabs from 231 women. Xpert GBS detected all of the cases (45, 19.5%), but 4 were missed by SCB. A rapid Xpert GBS service for women in labor would increase costs by ∼$55?000 per annum in our region.     

Development of conventional and real-time PCR assays for the rapid detection of group B streptococci

BACKGROUND: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. METHODS: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCycler(TM). For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. RESULTS: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only approximately 30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. CONCLUSION: These assays provide promising tools for the rapid detection and identification of GBS.     

Real-time quantitative PCR for the detection of Streptococcus pneumoniae in the middle ear fluid of children with acute otitis media

PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.     

B群链球菌免疫层析法的建立及其临床应用

目的建立一种B群链球菌(GBS)的胶体金免疫层析检测方法,并进行临床应用评价。方法收集202例阴道分泌物标本,采用细菌分离培养法作为金标准对标本的GBS进行检测,同时采用以双抗体夹心法为基础研制出B群链球菌胶体金免疫层析试纸条检测作为对照,分析其灵敏度、特异度,计算Kappa值分析两种方法的一致性。结果免疫层析法检测GBS的敏感度为97.50%,特异度为97.54%;与细菌分离培养法的总符合率为97.52%,Kappa值为0.948。结论免疫层析法检测GBS具有较高的敏感度和特异度,且简便准确,对GBS的筛查和快速诊断具有重要的临床应用价值。