肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

实时荧光RT-PCR检测活性单核细胞增生李斯特菌方法的建立

目的 采用逆转录结合实时荧光定量PCR技术,建立一种快速、准确、特异甄别单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李氏菌)死活状态的定量方法.方法 根据单增李氏菌hlyA基因序列设计引物和探针;对实时荧光PCR反应体系进行优化后,提取菌体mRNA,通过随机引物进行逆转录反应;产生的cDNA通过实时荧光定量PCR进行鉴定.进一步评价逆转录结合实时荧光定量PCR方法的特异性、灵敏度、重复性后,对20份模拟双盲样本进行检测.结果 本实验所建立的逆转录结合实时荧光定量PCR方法可准确、特异地检测单增李氏菌,其他菌株和失活的单增李氏菌均无阳性结果出现;该方法检测纯菌和模拟样本的灵敏度分别可达到10 CFU/ml和1000CFU/ml;定量检测的批间和批内的变异系数均小于5%;对20份模拟样本进行检测,其中10份含有活性单增李氏菌样本的检测结果均为阳性,其他含有失活单增李氏菌的5份样本和其他致病菌的5份样本检测结果为阴性.结论 本文建立的检测活性单增李氏菌实时荧光定量PCR方法快速、准确,结果可靠,实用性强,可进行定量分析,为食品安全监测和现场流行病学调查提供较好的分析手段和完整的数据.     

建立一种实时荧光PCR对维生素D受体基因单核苷酸多态性快速分型

目的:建立实时荧光PCR快速检测维生素D受体(VDR)基因rs2228570、rs1544410、rs7975232位点多态性。方法:以VDR基因的rs2228570、rs1544410、rs7975232三个SNP位点的变异碱基分别设计并合成特异性引物。11例体检健康儿童血液标本作为检测样本,采用实时荧光PCR方法检测样本中VDR基因型,采用Sanger法对检测结果测序验证。结果:利用所设计的特异性引物对人全血基因组中VDR进行SNP位点特异性扩增,根据7500 FAST实时荧光PCR仪的分析结果得到基因型,与测序结果比对后发现,11个样本采用实时荧光PCR方法基因分型结果与测序结果均一致。结论:实时荧光PCR快速检测VDR基因rs2228570、rs1544410、rs7975232位点多态性的方法或可用于临床VDR基因SNP快速分型。     

实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌方法的建立和应用

目的建立丙型副伤寒沙门菌和猪霍乱沙门菌的实时荧光PCR快速检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的丙型副伤寒沙门菌和猪霍乱沙门菌的保守序列,设计引物和改良分子信标探针,建立实时PCR检测方法。结果检测体系灵敏度高,纯DNA和菌液的最低检出限分别可达10fg和20CFU/反应体系;特异性好,对71株细菌的检测符合率达100%。20株沙门菌采取盲号模拟血培养标本进行血培养检测及鉴定,检出5株丙型副伤寒沙门菌和4株猪霍乱沙门菌,与试验的菌株相符。70份食品中用实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌均为阴性,而用传统方法分离培养未检出。结论建立的实时PCR检测方法可以快速、特异、灵敏地检测出丙型副伤寒沙门菌和猪霍乱沙门菌。     

耐甲氧西林葡萄球菌荧光定量PCR检测方法的实验研究

目的建立一种简便、特异的mecA基因荧光定量PCR检测方法，用于耐甲氧西林葡萄球菌（MRS）的快速鉴定。方法以煮沸法快速制备DNA模板，采用SYBRGreenI随机参入法，建立mecA基因的实时荧光定量PCR检测体系。并对检测体系的敏感性、特异性和灵敏度进行评价。结果本法对纯菌落的检测敏感性和特异性分别为98．5％和96．9％，检测灵敏度可达10^1CFU／ml，最小检菌量约为3个菌／反应体系。结论本实验所设计的荧光定量PCR方法用于MRS的检测具有快捷、高敏感性、高特异性和高灵敏度的特点。适用于MRS的快速检测。     

rpoB作为蜡样芽胞杆菌群快速检测的标志基因的实验研究

目的探讨常规PCR和实时荧光定量PCR（Q-PCR）方法检测蜡样芽胞杆菌群rpoB基因的特异性和敏感性。方法提取蜡样芽胞杆菌群和其他各种对照细菌的基因组DNA,合成蜡样芽胞杆菌群rpoB基因扩增引物,采用常规PCR和SYBRgreen实时定量PCR两种方法扩增rpoB基因片段,并将PCR产物克隆到pMD18-T载体后进行DNA测序。结果常规PCR和Q-PCR均能扩增出蜡样芽胞杆菌群rpoB基因的174bpDNA片段,而各种对照菌株均未见扩增。序列比对发现蜡样芽胞杆菌群细菌在该片段中存在5处核苷酸的不同,差异率为2.88%。以炭疽芽胞杆菌基因组DNA系列稀释作为扩增模板显示常规PCR最小检出量为3.42pg,Q-PCR的敏感性达到171fg,3次重复实验显示Q-PCR检测rpoB基因的灵敏度为（3.32×101±7.45×100）拷贝。结论以rpoB基因为检测靶基因的Q-PCR方法具有高度的特异性和良好的敏感性,能实现对蜡样芽胞杆菌群快速而准确的检测。     

实时荧光定量PCR在检测人ANP基因表达的应用

目的建立心房钠尿肽（ANP）基因mRNA表达水平的实时荧光定量PCR检测方法,并对该方法进行初步评价。方法以基因表达产物为模板,建立实时荧光定量PCR检测方法,对样本中的心房钠尿肽含量进行相对定量,比较不同样本组的基因表达水平。结果所建立的实时荧光定量PCR方法熔解曲线中熔解峰单一。肺癌患者胸腔样本的ANP含量为对照样本的4.68倍,血清样本为对照样本的16.03倍。结论所建立的ANP实时荧光定量PCR检测方法具有较高的特异性。肺癌患者胸腔液和血清中ANP的含量明显增高。     

空肠弯曲菌共同抗原单克隆抗体的研制及其在细菌检验中应用的研究

应用B-淋巴细胞杂交瘤技术,建立了5株稳定分泌抗空肠弯曲菌共同抗原(CA)单克隆抗体(McAb)的杂交瘤细胞株。其中4株的McAb针对CA中的耐热抗原成分,1株的McAb针对CA中的不耐热抗原成分。应用32种其它细菌对这些McAb的特异性检查表明,除与幽门弯曲菌发生交叉反应外,与其它细菌均为阴性反应。应用不同地区、不同来源的516株空弯菌对这些Mc-Ab与空弯菌的反应性进行了检查,结果均为阳性反应。应用BA-ELISA对人(140)、     

α变形菌纲磁螺菌属TaqMan探针实时荧光定量PCR快速检测方法的建立

目的建立一种快速有效检测环境中q变形菌纲磁螺菌属细菌的方法。方法本研究以α变形菌纲磁螺菌属保守且特异的16SrRNA片段为靶序列,化学合成基因序列,制备重组质粒作为磁螺菌属检测的标准品;用实时荧光定量聚合酶链反应（FQ—PCR）技术,针对目的片段基因设计特异性引物和TaqMan荧光探针,进行实时荧光定量PCR检测,运用统计学方法评价该方法的特异性、敏感性及重复性。结果本实验成功构建了质粒PUC19-QC,引物、探针特异性良好,标准曲线在5．10×10^1～5．10×10^7拷贝数之间具有较好的线性关系,相关系数R2=0．999。该法最低可检测到5．10x10个DNA拷贝数,灵敏度明显高于普通PCR;重复性试验表明．荧光定量PCR检测结果Ct值波动范围较小,α值的标准差均小于0．23,表明该法重复性好,可靠性高。析因方差分析表明不同稀释度之间的ct值差异有统计学意义（F=3125．305,P〈0．001）,三个批次的再现性良好,差异无统计学意义（F=0．057,P=0．945）,而浓度分组与时间之间也无交叉效应（F=0．533,P=0．873）。结论本实验所建立的TaqMan探针实时荧光定量PCR检测技术,能够快速有效地检测仅变形菌纲磁螺菌属细菌,且检测结果稳定,受外界条件如时间、气温等影响小。     

实时荧光PCR直接检测样本中耐甲氧西林金黄色葡萄球菌应用研究

目的 探讨实时荧光PCR直接检测病人样本中耐甲氧西林金黄色葡萄球菌的应用价值.方法 对80例经VITEK2COMPACT药敏鉴定系统鉴定为有金黄色葡萄球菌生长(其中MRSA40例,MSSA40例)及20例未检出金黄色葡萄球菌的患者样本进行实时荧光PCR扩增,检测样本中金黄色葡萄球菌特异性核酸片段(耐热核酸酶基因nuc)及耐药基因(mecA)并与药敏鉴定比较,判断实时荧光PCR法检测MRSA的敏感性及特异性;对于两种方法检测结果不一致的18例样本重新接种培养,收集金黄色葡萄球菌菌株进行实时荧光PCR扩增,检测nuc基因及mecA基因,以期发现两种方法检测结果差异原因.结果 100例临床样本两种方法检测结果一致共82例,其中MRSA一致40例,MSSA一致22例,非金葡20例;两种方法检测结果不一致18例,均为PCR基因检测为MRSA而药敏鉴定为MSSA.MRSA检测药敏鉴定阳性40例,检出率为40％,PCR基因检测阳性58例检出率为58％;PCR基因检出率明显高于药敏鉴定,二者结果差异有显著性(P＜0.05);以药敏鉴定为标准,PCR基因检测MRSA的敏感性为100％,特异性为70％;18例金黄色葡萄球菌菌株实时荧光PCR检测MRSA结果与药敏鉴定一致.结论 实时荧光PCR直接检测患者样本中MSSA具有高度敏感性及特异性,但检测MRSA会受到样本中其他含有mecA基因菌的干扰,对于临床结果解释需慎重.即便如此,该方法对于MRSA检测在院感防控方面仍有重要意义.     

Simultaneous detection of six diarrhea-causing bacterial pathogens with an in-house PCR-luminex assay

Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 10(3) to 10(5) CFU/g of stool for each pathogen as well as quantitative detection up to 10(9) CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (C(T)) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples.

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

Development and evaluation of immunochromatographic assay for simple and rapid detection of Campylobacter jejuni and Campylobacter coli in human stool specimens

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA, they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension, and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens, suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent, and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens.     

Use of the duplex TaqMan PCR system for detection of Shiga-like toxin-producing Escherichia coli O157

Real-time PCR assays have been applied for the detection and quantification of pathogens in recent years. In this study two combinations of primers and fluorescent probes were designed according to the sequences of the rfb(Escherichia coli O157) and stx2 genes. Analysis of 217 bacterial strains demonstrated that the duplex real-time PCR assay successfully distinguished the Escherichia coli O157 serotype from non-E. coli O157 serotypes and that it provided an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. On the other hand, bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for these two genes were linear for DNA concentrations ranging from 10(3) to 10(9) CFU/ml of E. coli O157:H7 in pure culture and milk samples. The real-time PCR allowed the construction of standard curves that facilitated the quantification of E. coli O157:H7 in feces and apple juice samples. The detection sensitivity of the real-time PCR assay ranged from 10(4) to 10(9) CFU/g (or 10(4) to 10(9) CFU/ml) for feces and apple juice and 10(5) to 10(9) CFU/g for the beef sample without enrichment. After enrichment of the food samples in a modified tryptic soy broth, the detection range was from 10(0) to 10(3) CFU/ml. The real-time PCR assays for rfb(E. coli) (O157) and stx2 proved to be rapid tests for the detection of E. coli O157 in food matrices and could also be used for the quantification of E. coli O157 in foods or fecal samples.     

Evaluation of culture methods and a DNA probe-based PCR assay for detection of Campylobacter species in clinical specimens of feces

Campylobacter species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for Campylobacter species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37 degrees C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (n = 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in Campylobacter spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis. Campylobacter species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically Campylobacter jejuni, C. concisus, C. curvus, and C. gracilis DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of Campylobacter species in fecal specimens. The finding of Campylobacter spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.     

Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR

We describe rapid PCR-biprobe identification of Campylobacter spp. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.     

Evaluation of a PCR/DNA Probe Colorimetric Membrane Assay for Identification of Campylobacter spp. in Human Stool Specimens

DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the Campylobacter genus, Campylobacter jejuni, and Campylobacter coli in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for C. jejuni. The assay is rapid and simple and can be applied to stool specimens for the detection of Campylobacter.     

Detection of Campylobacter spp. in chicken fecal samples by real-time PCR

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.     

Detection of Campylobacter species by using polymerase chain reaction and nonradioactive labeled DNA probe]

We have detected Campylobacter species which are now recognized as major pathogens of acute diarrheal disease in humans using polymerase chain reaction (PCR) and a nonradioactive labeled DNA probe. Diagnosis of Campylobacter enteritis without doing culture from stool samples is of great benefit in the laboratory. Two oligonucleotide primers (20 mer) complementary to a unique sequence of the DNA encoding ribosomal RNA (rRNA) of Campylobacter jejuni for PCR were synthesized by solid-phase phosphoamidite method. Amplified target DNA of 275 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with an oligodeoxynucleotide probe (28 mer) conjugated to alkaline phosphatase. In identification experiments, it was shown that the nonradioactive probe was hybridized to clinical strains of C. jejuni (104), C. coli (5), C. laridis (5), C. hyointestinalis (1) and C. fetus subsp. fetus (1) with an accuracy of 99-100%, while it was not for Helicobacter pylori. Further, there was no evidence of amplification in strains of K. pneumoniae, S. marcescens and E. coli. Using direct detection to stool specimens, this method could be performed in C. jejuni in 39 of 43 culture-positive specimens (91%), and in 19 of 141 culture-negative specimens (13.5%), respectively. The results of this comparative study suggested that the DNA probe assay became a rapid and reliable technique to confirm culture of Campylobacter species.