绿色魏斯氏菌实时荧光定量聚合酶链式反应检测方法的建立

以rpoA基因为靶基因,建立绿色魏斯氏菌SYBR Green Ⅰ实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）快速检测方法。针对rpoA基因设计特异性引物,建立绿色魏斯氏菌实时荧光定量PCR检测体系,通过特异性、灵敏度和重复性实验评价体系的检测效果,同时与常规PCR方法进行比较。结果表明：实时荧光定量PCR方法能够特异性检出绿色魏斯氏菌,对基因组DNA的检测灵敏度达到2.667×10－3 pg/μL,对纯培养物和模拟污染牛肉样品直接检测的灵敏度分别为30 CFU/mL和0.8 CFU/g;与常规PCR相比,实时荧光定量PCR检测的灵敏度是其1 000 倍;不同浓度样品独立重复实验循环阈值的标准差均小于1,变异系数在0.02%～1.28%之间。本研究所建立的绿色魏斯氏菌实时荧光定量PCR检测方法具有特异性好、灵敏度高、重复性好的特点,能够进行准确的定量检测,是快速检测绿色魏斯氏菌的有效手段。     

含内标的多重实时PCR法同时检测空肠弯曲菌和结肠弯曲菌

目的建立含内标的多重实时荧光PCR法同时检测空肠弯曲菌和结肠弯曲菌。方法针对空肠弯曲菌特有hipO基因和结肠弯曲菌特有ceuE基因设计引物探针,设计并优化内标DNA添加量。测试了方法的特异性、灵敏度以及在鸡肉中的检出限。结果内标的最适添加量为10~4copies/PCR。所建立方法对空肠弯曲菌和结肠弯曲菌的灵敏度分别达到4.7copies/PCR和5.23copies/PCR;对115株空肠弯曲菌、49株结肠弯曲菌和42株非目标菌株在3种不同类型的实时荧光PCR仪上的特异性均达到100%;对鸡肉中空肠弯曲菌和结肠弯曲菌的检出限达到10CFU/25g,与传统检测方法一致。采用所建立的方法对50份市售生鲜鸡肉进行检测发现,空肠弯曲菌阳性率为12%(6/50),结肠弯曲菌阳性率为4%(2/50);传统国标检测方法除了1份空肠弯曲菌阳性样品未得到分离确认,其余PCR阳性样品均在平板上分离确认。结论该方法特异性强、灵敏度高、开放性好、含有内标可防止"假阴性",可应用于食品中2种重要致病性弯曲菌的快速同步检测。     

RPA等温扩增技术检测副溶血性弧菌

目的:建立副溶血性弧菌的RPA-exo荧光探针快速检测方法。方法:采用重组酶聚合酶扩增技术,以irgB为靶基因设计副溶血性弧菌的RPA-exo引物探针,对引物探针进行组合筛选,建立RPA-exo荧光探针快速检测方法,并对其特异性、灵敏度、模拟污染实验及实际应用效果进行测试。结果:建立的方法15 min可获得结果,具有高特异性,无交叉反应;灵敏度为1.0×103 CFU/mL,DNA检测限为0.35 pg/μL,质粒检测限为1×103 copies/μL;模拟污染实验中加入终浓度为1.36×103 CFU/mL时,无需增菌即可被检出;实际样品检测中,10份水产品,有3份样品被检出,检测结果与国标GB 4789.7-2013结果一致。结论:本研究成功建立了副溶血性弧菌的RPA-exo快速检测方法。     

大肠杆菌O157:H7特异基因的实时荧光定量PCR检测

为建立快速、特异的检测大肠杆菌O157:H7的实时荧光定量聚合酶链式反应(real time polymerase chainreaction,RT-PCR)方法,针对大肠杆菌O157:H7的特异基因rfbE设计一对特异引物,建立SYBR GreenⅠ实时定量PCR检测方法,并进行灵敏度、重复性和特异性实验,同时与常规PCR方法进行比较。结果显示所建立的SYBRGreenⅠ实时定量PCR方法可以快速、特异地检测出大肠杆菌O157:H7,细菌纯培养物中其灵敏度可达2×101CFU/mL,临床模拟污染肉样中能最低能检测到1×102CFU/mL的大肠杆菌O157:H7。与常规PCR方法相比,SYBR GreenⅠ实时定量PCR方法对临床样品中大肠杆菌O157:H7的检出率大大提高。本研究建立的SYBR GreenⅠ荧光定量PCR技术能快速准确、特异、敏感地检测大肠杆菌O157:H7。     

婴幼儿奶粉中阪崎肠杆菌双重荧光PCR快速检测方法的建立

旨在建立针对阪崎肠杆菌的双重荧光PCR快速检测方法。以阪崎肠杆菌局部大分子合成(MMS)操纵子和外膜蛋白A(ompA)为靶基因,建立双重荧光PCR反应体系,探讨该体系的特异性、灵敏度和抗干扰能力。结果表明,双重荧光PCR体系对阪崎肠杆菌的灵敏度为4.3×103 CFU/mL,人工污染初始菌量为2 CFU/100 g奶粉样品增菌24 h即可检出;39株实验菌中的15株阪崎肠杆菌出现特异性扩增,24株非阪崎肠杆菌未出现特异性扩增。本研究所建立的双重荧光PCR体系特异好、灵敏度较高及抗干扰能力强,可用于婴幼儿奶粉中阪崎肠杆菌的快速检测。     

多重PCR快速检测两种致泻性大肠杆菌方法的建立

本研究以肠出血性大肠杆菌O157∶H7及肠侵袭性大肠杆菌为目标菌,针对大肠杆菌共同基因uid A、肠出血性大肠杆菌O157∶H7的特异基因O-antigen、肠侵袭性大肠杆菌的特异基因ipa H设计3对引物,建立并优化了检测两种菌的多重PCR检测方法,进行了特异性验证和灵敏度分析,并应用于人工接种新鲜莴苣的检测中。实验结果表明,本研究建立的三重PCR快速检测两种致病菌的检出限为6.3×103CFU/m L,具有较好的灵敏度和特异性。利用所建立的多重PCR方法对人工接种肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌的新鲜莴苣进行检测,检出限为7.8×104CFU/m L。此方法能够对肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌两种食源性致病菌进行快速检测。     

环介导等温扩增技术快速检测肉中沙门氏菌

应用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立了一种快速、高效、灵敏的肉中沙门氏菌的检测方法。针对沙门氏菌的属特异性基因inv A设计2对引物,对人工污染肉样以及实际肉样分别进行检测。结果表明:所建立的LAMP反应能够特异性的检测沙门氏菌,对沙门氏菌纯菌的检测灵敏度为普通PCR的100倍,可达到9.8×100CFU/m L。LAMP检测人工污染沙门氏菌肉样的检测限为9.8×101CFU/m L。因此,本实验利用LAMP建立的沙门氏菌检测方法具有快速、灵敏、特异、操作简便的特点,具有广泛发展前景。     

食品中荧光假单胞菌双重PCR法检测体系的建立和评价

根据荧光假单胞菌(Pseudomonas fluorescens)促旋酶(gyrase)的B亚单位基因以及金属蛋白酶(apr)基因设计出两对引物,经过反应条件的优化,建立了用于荧光假单胞菌快速检测的双重聚合酶链式反应(PCR)方法,并对该体系进行评价。结果显示,Pseudomonas fluorescens菌株经普通PCR扩增均可见2条特异性条带(384 bp和194 bp),而其它阴性对照菌株PCR扩增均为阴性。基于基因组DNA的双重PCR检测灵敏度为16.9 fg/μL(3~4拷贝/μL),1.8CFU/m L的UHT牛奶样品(250 m L)经增菌24 h后用该双重PCR方法均可检出。本研究建立的双重PCR检测方法具有良好的特异性和灵敏度,能克服乳制品样品基质的干扰,可应用于乳制品中荧光假单胞菌的快速检测。     

预包装柳州螺蛳粉沙门氏菌实时荧光PCR快速检测

目的建立实时荧光PCR法快速检测预包装柳州螺蛳粉中沙门氏菌的分析方法。方法根据沙门氏菌invA基因设计引物和探针,优化反应体系中的探针浓度后对人工添加沙门氏菌和干扰菌模拟受污染的预包装柳州螺蛳粉样品进行检测。结果设计的引物和探针只对阳性菌株有荧光反应,具有良好的特异性,最佳反应探针终浓度为0.2μmol/L,检测灵敏度为100CFU/mL,扩增效率103.92%;对模拟污染的预包装柳州螺蛳粉经过一步增菌18 h后最低能检测出4 CFU/25 g沙门氏菌。结论实时荧光PCR检测灵敏度高、特异性强,可应用于预包装柳州螺蛳粉中沙门氏菌的快速高效检测。     

Quantification and differentiation of Campylobacter jejuni and Campylobacter coli in raw chicken meats using a real-time PCR method

Campylobacter species are one of the most common causes of bacterial diarrhea in humans worldwide. The consumption of foods contaminated with two Campylobacter species, C. jejuni and C. coli, is usually associated with most of the infections in humans. In this study, a rapid, reliable, and sensitive multiplex real-time quantitative PCR was developed for the simultaneous detection, identification, and quantification of C. jejuni and C. coli. In addition, the developed method was applied to the 50 samples of raw chicken meat collected from retail stores in Korea. C. jejuni and C. coli were detected in 88 and 86% of the samples by real-time quantitative PCR and the conventional microbiological method, respectively. The specificity of the primer and probe sets was confirmed with 30 C. jejuni, 20 C. coli, and 35 strains of other microbial species. C. jejuni and C. coli could be detected with high specificity in less than 4 h, with a detection limit of 1 log CFU/ml by the developed real-time PCR. The average counts (log CFU per milliliter) of C. jejuni or C. coli obtained by the conventional methods and by the real-time PCR assay were statistically correlated with a correlation coefficient (R2) between 0.73 and 0.78. The real-time PCR assay developed in this study is useful for screening for the presence and simultaneous differential quantification of C. jejuni and C. coli.     

双重PCR 方法检测沙门氏菌和空肠弯曲菌

为建立能够同时检测食品中沙门氏菌和空肠弯曲菌的双重PCR 方法。采用沙门氏菌鞭毛基因fimY 和空肠弯曲菌马尿酸酶基因hipO 设计特异性引物,并对影响PCR 扩增的主要因素——引物浓度、退火温度、Mg2+ 浓度因素进行优化,比较单一PCR 和双重PCR 的检测效果。结果表明：采用单一PCR 法检测沙门氏菌和空肠弯曲菌时,灵敏度分别可达到3.98pg 和4.05pg;而采用双重PCR 检测时,灵敏度较单一PCR 法有所下降,沙门氏菌和空肠弯曲菌检出限量分别为398pg 和40.5pg。本研究建立的特异性强和灵敏度高的双重PCR 检测方法,可为实现食品中沙门氏菌和空肠弯曲菌的同时检测提供新方法。     

铜绿假单胞菌LAMP快速检测方法建立及微滴数字PCR验证

目的:采用环介导等温扩增技术建立铜绿假单胞菌快速检测方法,并用微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)技术进行方法验证。方法:以铜绿假单胞菌rpod基因为靶基因设计引物,建立了果汁饮品铜绿假单胞菌快速检测方法,对于方法的可靠性、特异性和灵敏度,采用ddPCR验证。结果:本实验建立的LAMP快速检测方法显色结果明显,ddPCR检测,以铜绿假单胞菌悬液浓度为横坐标,以数字PCR扩增copy数为纵坐标,生成标准曲线y=0.07497x-9.3516,线性关系良好,R2=0.9999。铜绿假单胞菌检测范围1.5×102~1.5×105 CFU/mL;方法灵敏度高、特异性强,标准菌株最低浓度为1 ng/μL;标准差分别为0.57、0.71、4.24、14.8,RSD值在0.66%~22.8%之间。检测果汁饮品128份,126份未显色,显色样品2份,以标准曲线定值,浓度分别为1.64×104、2.29×102 CFU/mL。     

荧光免疫层析法结合免疫磁珠分离技术快速检测单增李斯特菌

为建立单增李斯特菌简单快速、灵敏度高和特异性强的检测方法,本研究以抗单增李斯特菌单克隆抗体偶联磁珠制备免疫磁珠;以羧基荧光微球标记的抗单增李斯特菌多克隆抗体及鼠IgG为标记抗体,抗单增李斯特菌多克隆抗体和羊抗鼠二抗分别作为检测线和质控线制备荧光免疫层析试纸条。将免疫磁珠分离与荧光免疫层析法相结合应用于单增李斯特菌的现场快速检测中。结果表明:荧光免疫层析试纸条对纯培养单增李斯特菌的检测限为4×105CFU/mL,联合检测方法10倍、100倍浓缩时,检测限分别为4×104CFU/mL和1×104CFU/mL。联合检测体系特异性较好,与实验室保存的10株细菌无交叉反应。人工污染样本检测限为1×104CFU/mL,同纯培养物相比检测灵敏度并没有降低。本方法的建立对于食品中单增李斯特菌的现场快速检测具有重要意义。     

Detection of Campylobacter jejuni and Campylobacter coli in foods by enrichment culture and polymerase chain reaction enzyme-linked immunosorbent assay

A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.     

Rapid detection of Campylobacter jejuni in chicken rinse water by melting-peak analysis of amplicons in real-time polymerase chain reaction

Five DNA extraction protocols for the detection of Campylobacter spp. by polymerase chain reaction (PCR) were compared. A method involving Triton X-100 produced template DNA of sufficient quality to allow the detection of Campylobacter jejuni at levels of 100 CFU/ml in pure culture. Primers were designed on the basis of the cadF gene sequence. With a SYBR Green I real-time PCR assay, these primers amplified only sequences present in C. jejuni to produce a product with a melting temperature of 81.5 degrees C. None of the strains of Campylobacter coli, Campylobacter lari, or Campylobacter fetus tested produced this product during the PCR assay. Other noncampylobacter species tested were shown not to possess the cadF sequence. The real-time PCR combined with a rapid, simple Triton X-100 DNA extraction protocol made it possible to detect < 10 CFU of C. jejuni per ml of chicken rinse within 14 h.     

Isolation and polymerase chain reaction-based detection of Campylobacter jejuni and Campylobacter coli from poultry in the Philippines.

The polymerase chain reaction (PCR) and the conventional culture method of detecting thermophilic Campylobacter species in duck and chicken samples from two locations in the province of Laguna, Philippines, were compared. Three Campylobacter jejuni and five C. coli strains were isolated from a total of 135 duck and chicken samples from both methods. The PCR technique, however, was found to be more sensitive, accurate and rapid than the conventional culture method. The specificity of two sets of published primers, C442-C490 (specific for C. jejuni, C. coli and C. lari) and CL2-CR3 (specific for C. jejuni) were confirmed with reference and field strains. To improve detection, a lysate was prepared by boiling cells in Triton X-100, and then used as template for PCR to detect Campylobacter from spiked and naturally contaminated chicken rinse. For spiked chicken samples, a 17-h Meuller-Hinton Broth enrichment for the chicken rinse resulted in an improved sensitivity at 31.7 CFU/g using C442-C490. This enrichment-PCR tandem also detected thermophilic Campylobacter from 1 out of 21 native chicken samples from a wet market. To our knowledge, this is the first report of thermophilic Campylobacter isolation from poultry in the Philippines. The approaches described here could serve as a basis for future surveillance and/or epidemiological studies on this emerging foodborne pathogen.     

Detection of Campylobacter jejuni in naturally contaminated chicken skin by melting peak analysis of amplicons in real-time PCR

Contamination of poultry by Campylobacter spp. is a significant source of human diarrheal diseases. Traditional methods currently used to detect Campylobacter in foods are time-consuming and labor-intensive. In this study, primers designed for the Campylobacter jejuni cadF gene sequence were used in a SYBR Green I real-time PCR assay as an alternative to a conventional bacteriological method for the rapid detection of C. jejuni from poultry. Twelve portions of chicken purchased from two local grocery stores and 39 portions obtained from a commercial processing plant were examined. Samples of the skin were enriched in Bolton broth at 37 degrees C for 3 h and then at 42 degrees C for 9, 21, or 45 h under microaerobic conditions. DNA was extracted from 1-ml aliquots of the enrichment cultures using 1% Triton X-100. The DNA was used as the template in a real-time polymerase chain reaction (PCR) assay. After 24 h of enrichment, C. jejuni was isolated from 13 samples and all of the positive cultures were also detected by the real-time PCR procedure. C. jejuni was detected by both methods from samples artificially contaminated with 1 or 10 CFU of C. jejuni per 10 g, after 24 h of enrichment. The real-time PCR method was found to be sensitive and specific. It significantly reduced the time required for the detection of C. jejuni in poultry following enrichment of samples.     

食源性MRSA双色荧光PCR检测方法建立

目的建立快速检测食源性耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)Taqman探针双色荧光PCR方法。方法根据金黄色葡萄球菌种属鉴定nuc基因和MRSA决定因子mec A基因,设计合成引物探针,建立双色荧光PCR扩增体系。利用所建立的方法检测特异性及灵敏度。将金黄色葡萄球菌依次传代培养,检测不同代次的菌株验证方法的稳定性,并对实际样品分离株进行检测验证方法的可行性与实用性。结果该方法可准确并特异性检测出MRSA和甲氧西林敏感金黄色葡萄球菌(methicillin-susceptible Staphylococcus aureus,MSSA),检测MRSA的nuc基因和mec A基因的灵敏度可达2.7×103 CFU/m L,不同代次的菌株的检测结果一致。结论本实验所建立的双色荧光PCR检测方法具有良好的特异性、灵敏度及稳定性,可用于快速检测食源性MRSA。     

食品中沙门氏菌FTA膜结合跨越式滚环等温扩增检测方法的建立

为实现食品中沙门氏菌的简便和快速现场检测,本研究采用FTA膜(Flinders technology associates,FTA)结合跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)方法(FTA-SRCA)建立一种新型的沙门氏菌检测方法。利用FTA膜快速提取模板DNA,根据沙门氏菌的inv A基因设计及筛选引物,建立FTA-SRCA反应体系。扩增反应在能够实现集约化检测的凹孔板中进行,反应结束后添加荧光染料观察结果。确定了该方法的特异性、灵敏度和人工污染样品的检出限,并对60个实际样品进行检测,评估其敏感性、特异性和符合率。结果表明:检测的17株沙门氏菌均为阳性结果,29株非沙门氏菌均为阴性结果,特异性良好。FTA-SRCA方法的灵敏度为6.81×100 CFU/m L,比PCR方法高100倍,比SRCA方法高10倍。对于人工污染的牛奶样品检测,FTA-SRCA方法的检出限为3.22×100CFU/m L,比PCR方法低1000倍,比SRCA方法低10倍。检测实际样品的敏感性、特异性和符合率分别为100.00%,94.64%,95.00%。本研究建立的FTA-SRCA方法具有操作简便快速、成本低廉、特异性强、灵敏度高、检出限低等优点,可用于食品中沙门氏菌的大批量集约化快速现场检测。