牛奶中3种致病菌多重PCR的建立及初步应用

为了能同时在牛奶中快速检测金黄色葡萄球菌、大肠杆菌、沙门氏菌,本研究根据GenBank中的金黄色葡萄球菌femB、大肠杆菌phoA、沙门氏菌invA基因序列,分别设计3对特异性引物(目的片段分别为233 bp、444 bp、724 bp),通过对PCR反应条件的优化建立了多重PCR方法。敏感性、特异性试验结果显示,该多重PCR对金黄色葡萄球菌、大肠杆菌、沙门氏菌的最低核酸检测量分别为13.3 pg/mL、13.3 pg/mL、133.3 pg/mL,而对牛结核杆菌、多杀性巴氏杆菌、牛支原体、单核细胞增生李斯特氏菌、志贺氏菌的扩增结果均为阴性。对25分临床样品的检测结果表明,该多重PCR检测结果与单一PCR检测结果一致。结果表明,建立的多重PCR方法具有很好的特异性和敏感性,可用于金黄色葡萄球菌、大肠杆菌和沙门氏菌的快速鉴别诊断。     

乳中志贺氏菌的荧光定量PCR检测方法的建立

为了快速、有效的检测乳中的志贺氏菌,建立一种特异性强、灵敏度高的荧光定量PCR方法。根据GenBank公布的志贺氏菌ipaH基因的保守序列设计特异性引物,对提取的志贺氏菌DNA模板进行PCR扩增,对目的基因进行克隆和测序,然后利用荧光定量PCR方法,对志贺氏菌进行检测,确定其扩增条件。建立的方法特异性强,检测的灵敏度可达到10-6 ng/μL。利用建立的方法可检测乳中2.84 cfu/mL的志贺氏菌。该方法为快速、准确检测乳中的志贺氏菌提供了参考。     

4种食源性致病菌的多重PCR快速检测方法研究

为快速检测蜡样芽孢杆菌、金黄色葡萄球菌、志贺氏菌和沙门氏菌4种食源性致病菌,建立4重聚合酶链式反应(polymerase chain reaction,PCR)的检测方法。针对菌株的特异性基因设计引物,选择退火温度相近,扩增条带区间不同的引物为多重聚合酶链式反应引物组,并对多重PCR的引物浓度、退火温度进行优化,确定扩增条件。结果表明,在54℃的退火温度下,4种菌可扩增长度为246、166、65、107 bp的清晰片段,其检测灵敏度为蜡样芽孢杆菌2×101CFU/mL,金黄色葡萄球菌可达1.3×102 CFU/mL,志贺氏菌可达1.3×102 CFU/mL,沙门氏菌可达1.4×102 CFU/mL。     

检测食品中志贺氏菌的双抗夹心ELISA方法的研究

研究获得纯化抗志贺氏菌IgY,经检测10mg/mL纯化抗志贺氏菌IgY的效价为1∶320;以志贺氏菌免疫新西兰大耳白兔,获得抗志贺氏菌的兔抗体,效价可达1∶12800。以此两种抗体建立双抗夹心ELISA,正交试验分析表明,兔抗体包被条件为4℃过夜,采用不封闭,抗原与包被抗体结合条件为37℃、1h;加入检测抗体(IgY)的浓度为0.25mg/mL,其结合条件为37℃、1h。该方法对纯培养菌液检出限为105cfu/mL,具有良好的敏感性;10株其他菌株的检测结果表明,该方法只与志贺氏菌发生特异性反应,不与其他菌株的抗原发生交叉反应。染菌的食品样品经选择性增菌后进行双抗夹心ELISA检测,含0.1～1cfu/mL志贺氏菌的样品在增菌13h后可检出阳性反应,含1～10cfu/mL志贺氏菌的样品在增菌11h后可检出阳性反应。     

基于rpoB靶基因的PCR法快速检测弓形菌

建立了一种弓形菌(Arcobacter)快速、敏感、特异的PCR检测方法.采用PCR技术扩增弓形菌编码RNA聚合酶β亚基的rpoB基因,并对该方法进行特异性和敏感性测试.结果只有弓形菌在205bp处出现目的扩增带,其他菌株扩增结果均为阴性.该法对弓形菌的检测限可达8.20×102cfu/mL.建立的PCR方法具有操作简便、准确可靠以及灵敏特异的特点,为食品中弓形菌的快速检测提供了新的手段.     

传统发酵豆制品中3种致病菌的三重荧光PCR快速检测方法的建立

为了建立传统发酵豆制品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光PCR快速检测方法。以单增李斯特菌hly A基因、蜡样芽孢杆菌Cereolysin AB基因和金黄色葡萄球菌nuc基因为靶基因设计引物与TaqMan探针,通过优化PCR反应体系,建立了可同时检测单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光定量PCR体系,并进行了特异性和敏感性试验。结果显示,该方法灵敏度高,特异性强,重复性好。对26株非目标菌进行检测,结果均为阴性,而定量检测批内和批间的变异系数均小于2%。单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌敏感性试验结果表明,这三种细菌的最低检测浓度分别为3×103cfu/mL、2×104cfu/mL、2×104cfu/mL。应用该方法可在8h内完成对样品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的同步检测。     

大肠埃希O157:H7等致病菌多重PCR法快速检测试剂盒的研制

目的:研制一种能够同时快速检测大肠埃希O157:H7、志贺氏菌和致病性蜡样芽孢杆菌的三重PCR试剂盒.方法:以大肠埃希O157:H7的hlyAB基因、痢疾志贺氏菌的IpaH基因和致病性蜡样芽孢杆菌的hblA基因作为目的基因片段O157:H7、痢疾志贺氏菌和致病性蜡样芽孢杆菌的特异性抗原基因序列,分别设计1对引物,并对其反应条件进行优化,建立1种多重PCR检测试剂盒.结果:本试剂盒可准确检测出生肉和即食肉制品中的上述3种致病菌,O157:H7检出极限为19.8 cfu/mL,志贺氏菌检出极限为17 cfu/mL,蜡样芽孢杆菌检出极限为17.7 cfu/mL.可在5 h内完成全部反应过程,得出检测结果.结论:本试剂盒在理论和实际应用方面均具有优越性,能够同时检测上述3种病原菌,可用于食品及其原料的生物安全检测,也可用于兽医临床诊断.     

抗志贺氏菌IgY的提纯及建立间接ELISA检测志贺氏菌

采用水稀释法提纯抗志贺氏菌IgY,检测10mg/mL纯化抗志贺氏菌IgY的效价为1∶320,并以此IgY为基础建立间接ELISA检测志贺氏菌,测定志贺氏菌纯培养液的检出限为105～106cfu/mL。对蜡样芽孢杆菌等10株不同菌株的检测结果表明,该方法对志贺氏菌有明显的检测特异性,对所测定的其它菌株无交叉反应。     

PCR和实时PCR技术快速检测牛奶样品中的沙门氏菌

根据沙门氏菌组氨酸转运操纵子基因的保守序列设计特异性引物,建立快速检测牛奶中沙门氏菌的方法.比较不同的DNA模板制备方法,将快速常规FCR与实时PCR结合,对阳性培养液及牛奶中的沙门氏菌进行检测.结果表明,采用设计的引物能有效地扩增出沙门氏茵特有的大小为495 bp的基因片段,与GeneBank 上的序列大小一致.常规PCR菌液敏感度可达到8 cfu/mL,检出时间少于20 h.实时PCR的DNA模板最低可达1.59x10-5μg/μL,检测时间仅需3 h.新建的PCR检测方法准确、敏感、特异、快速.     

单增李斯特菌与志贺氏菌多重PCR检测技术的建立

目的:为食品、卫生行业提供一种简便、灵敏、快速,并且特异性强的单增李斯特菌(Listeria Monocyto-genes)与志贺氏菌(Shigella)多重PCR检测方法.方法:根据单增李斯特茵转录调节子基因prfA、志贺氏茵侵袭性质粒抗原H基因ipaH筛选出引物;优化多重PCR的引物浓度、退火温度及Mg2+等条件,分析单-PCR及多重PCR的特异性、灵敏度;结合24 h增茵培养.确定多重PCR检测人工污染脱脂灭茵乳的检出限.结果:多重PCR扩增出了预计的PCR产物,即单增李斯特茵(274bp)和志贺氏茵(421bp).该方法的灵敏度:78 pg单增李斯特茵基因组DNA、7.5pg志贺氏茼基因组DNA.多重PCR检测人工污染脱脂灭菌乳,单增李斯特茵与志贺氏茵的灵敏度分别为2cfu/25mL和1.6cfu/25mL.结论:该方法在食品、卫生行业具有很好的应用和开发前景.     

Saltatory rolling circle amplification for sensitive visual detection of Staphylococcus aureus in milk

Monitoring Staphylococcus aureus with high sensitivity is very important for ensuring milk quality and food safety. In this study, we used a rapid nucleic acid isothermal amplification method, saltatory rolling circle amplification (SRCA), for the detection of Staph. aureus in milk. The results of the SRCA method can be assessed visually by the presence of white precipitate or by fluorescence measurement. Thirteen Staph. aureus strains and 31 non-Staph. aureus strains were used to evaluate the specificity of SRCA. The method exhibited excellent detection of Staph. aureus genomic DNA at a concentration of 7.8 × 10¹ fg/µL when assessed by visible precipitate, and at 7.8 × 10⁰ fg/µL when detected by fluorescence after addition of the fluorochrome SYBR Green I. In artificially inoculated milk, the detection limits of SRCA were 5.6 × 10² cfu/mL by precipitate and 5.6 × 10¹ cfu/mL by fluorescence, respectively. Compared with conventional PCR approaches, the SRCA assay achieved at least 100-fold higher sensitivity. Moreover, the sensitivity, specificity, and accuracy of the SRCA-based system were calculated to be 100.00, 97.73, and 97.78%, respectively. These results indicate that SRCA has potential application as a sensitive and visual technique for the detection of Staph. aureus in milk.     

Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 10⁰ cfu/mL and 5.7 × 10¹ cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.     

食品中金黄色葡萄球菌核酸层析检测技术的研究

核酸层析检测技术是将PCR与免疫层析技术相结合,用于食品中金黄色葡萄球菌的快速检测。该技术根据金黄色葡萄球菌n犹基因序列,设计特异性的引物,分别标记生物素和异硫氰酸荧光素（FITC）,用胶体金免疫层析技术进行检测。通过12株金黄色葡萄球茵及26株常见致病菌的检测结果说明其特异性强,检测灵敏度达到5×10^3du／mL,样品检测限达到0．5efu／g,与传统方法（GB／T4789．10—2010）结果相比较,无显著性差异x^2=0,P〉0．05）,可以应用于食品中金黄色葡萄球菌的快速检测与鉴定。     

Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii,Bacillus cereus,and Salmonella spp. in milk and milk products

Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. are common food-borne pathogens. The aim of this study was to develop a sensitive, specific, and rapid method for the simultaneous detection of these 3 pathogens in milk and milk products. Three specific primers were designed based on ompA, invA, and cesB of C. sakazakii, Salmonella spp. and B. cereus, respectively, for use in a multiplex PCR (mPCR). To eliminate false-positive results, cells were pretreated with propidium monoazide (PMA) for the selective elimination of the genomic DNA of dead cells. An internal amplification control was applied as an indicator of false-negative results from the interference of inhibitors in the food matrix. Results showed that, in pure culture, the limits of detection of the assay for C. sakazakii, Salmonella Enteritidis, and B. cereus were 9.5 × 10⁴, 7.4 × 10², and 7.5 × 10² cfu/mL, respectively. Moreover, 8 cfu/mL of viable B. cereus cells were detected after 5 h of enrichment, and 9 cfu/mL of viable C. sakazakii and 7 cfu/mL of Salmonella Enteritidis were detected after 7 h of enrichment in spiked pure milk, walnut peanut milk, and whole-wheat milk. To validate the PMA-mPCR assay, the PMA-mPCR assay and the traditional culture method were performed to detect the 3 bacterial strains in 1,165 milk product samples. The PMA-mPCR assay obtained the same results as the culture-based method. Results demonstrated that the PMA-mPCR assay has excellent sensitivity and specificity for the simultaneous detection of viable C. sakazakii, Salmonella Enteritidis, and B. cereus in milk and milk products.     

环介导等温扩增方法在食品中沙门菌检测的应用和评价

将环介导等温扩增检测方法应用于食品中沙门菌的检验,并在检测方法特异性、灵敏度等方面与实时荧光PCR和传统检测方法进行比较。方法 针对沙门菌属高度保守的fimY基因设计环介导等温扩增检测引物并优化反应体系,在特异性、灵敏度和实际样品检测等方面与实时荧光PCR及传统检测方法比对。结果 本研究建立的LAMP方法检测沙门菌93株和非目标菌31株,具有良好的特异性。在纯培养、无需增菌情况下,其检测灵敏度为6.4×10²cfu/ml,与实时荧光PCR方法相当。食品基质添加试验中,环介导等温扩增方法检测低限为2cfu/25g样品;对45份实际食品样品检测结果表明,该方法实际样品检出率为11.1%,与实时荧光PCR及传统方法检测结果一致。结论 本研究建立的沙门菌环介导等温扩增检测方法具有良好的特异性,检测灵敏度与实时荧光PCR相当,适用于沙门菌的快速筛选。     

环介导等温扩增技术快速检测奶粉中的单增李斯特菌

环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一种在等温条件下特异、灵敏、快速的新型基因扩增技术。试验以单增李斯特菌为研究对象,根据其特有的hlyA基因设计了一套特异性引物,进行LAMP扩增。结果表明,LAMP检测单增李斯特菌的灵敏度为2.45×101cfu/mL,其对人工感染奶粉样品的检出限为7.32×101cfu/mL。对其它食品病原菌进行检测,结果均未出现目的条带,特异性强。表明LAMP法适合于食品中污染单增李斯特菌的快速检测。     

Rapid and simple quantitative identification of Listeria monocytogenes in cheese by isothermal sequence exchange amplification based on surface-enhanced Raman spectroscopy

Foodborne pathogens detection is important to ensure food safety and human health. In this study, we designed a comet structure to rapidly and sensitively detect foodborne Listeria monocytogenes. This method combined isothermal sequence exchange amplification (SEA) and surface-enhanced Raman spectroscopy. Listeria monocytogenes DNA could be rapidly amplified at a constant temperature via SEA with a pair of modified primers, which rendered the precise thermal control instrumentation unnecessary. Efficient SEA amplification generated a large number of DNA duplexes that could be easily captured by streptavidin-modified magnetic bead and Au^MB@Ag-isothiocyanate fluorescein antibody (anti-FITC). Au^MB@Ag-anti-FITC was used as a signal probe, which generated a significant excitation signal at 1,616 cm^?1 for quantitative detection and analysis. The results displayed sensitive detection of L. monocytogenes in cheese from 2.0 × 10¹ cfu/mL to 2.0 × 10⁶ cfu/mL within 1.0 h with a detection limit of 7.8 cfu/mL. Furthermore, this comet structure displayed the desirable specificity as its specific primers and amplified DNA ends were attached to streptavidin-modified magnetic beads and Au^MB@Ag-anti-FITC, respectively. We expected that the method devised would provide a promising new approach to screening for L. monocytogenes and guarantee the microbiological safety of dairy products.     

环介导等温扩增检测猪血中金黄色葡萄球菌

采用环介导等温扩增(LAMP)技术,检测猪血中金黄色葡萄球菌.以金黄色葡萄球菌(CMCC10201)的femA基因作为靶序列,设计LAMP和PCR引物,通过凝胶电泳,判断检测结果.对8株常见致病菌进行LAMP特异性实验,表明对金黄色葡萄球菌的检测具有很高的特异性;LAMP检测金黄色葡萄球菌的灵敏度为8.7CFU/mL,直接检测猪血中金黄色葡萄球菌的检出限为8.7×102CFU/mL,PCR法的检出限为8.7×103CFU/mL.本实验所建立的快速检测猪血中金黄色葡萄球菌的LAMP法具有较高的特异性和敏感性,能够满足快速检测的需要.     

IMS USING IN-HOUSE MONOCLONAL ANTIBODY-COATED MAGNETIC BEADS ASSOCIATED TO PCR ASSAY FOR DETECTION OF SALMONELLA TYPHIMURIUM IN RAW MEATS

An immunomagnetic separation (IMS) technique and a PCR assay were developed for use in detection of Salmonella Typhimurium in meat samples. To prevent false-negative results, an internal amplification control was developed. The polymerase chain reaction (PCR) using primers specific for an omp gene sequence of Salmonella spp has shown 100% sensitivity and specificity and a detection limit of 10⁴ cfu/mL. The IMS-PCR methods using PCR immediately after IMS and using 6 h postenrichment in brain heart infusion between IMS and PCR resulted in detection limits of 10³ cfu/mL and 1–10 cfu/mL, respectively. The lowest level of S. Typhimurium that could be detected by the IMS-PCR method in the presence of natural microbiota from inoculated meat samples was 1–10 cfu/25 g. When samples were analyzed using enrichment protocols without IMS, several false-negative results were obtained.

PRACTICAL APPLICATIONS

The immunomagnetic separation-polymerase chain reaction (IMS-PCR) method developed enabled a rapid and sensitive detection of Salmonella Typhimurium in inoculated meat samples. Monoclonal antibody (Mab)-coated magnetic beads prepared in-house were efficient in concentrating and separating the bacteria from the food matrix, thus improving detection limit and avoiding false-negatives. The internal amplification control (IAC), now mandatory in PCR assays, using the same primers of the target DNA further prevented false-negative results. Therefore, the IMS-PCR method developed in this study could be used in the future by the Brazilian food industry as a substitute for the expensive imported kits for Salmonella detection in foods. We are now developing a panel of Mabs against conserved antigens of Salmonella for use in the IMS-PCR method in order to extend its applicability for detection of other serovars.     

Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria

The efficient and timely detection of pathogens is a major concern worldwide. The aim of this study was to establish a rapid detection method for Salmonella bacteria in food samples to facilitate timely treatment. Widely used detection methods currently include culture-based methods and PCR-based methods. The former are time consuming, requiring 2 to 3 d, whereas the latter have higher accuracy but are typically complicated, requiring expertise and expensive instruments. In this study, a sensitive and rapid approach for the visual and point-of-use detection of Salmonella bacteria based on recombinase polymerase amplification (RPA) and a lateral-flow (LF) nucleic acid strip was established. We designed a pair of primers according to the invA gene of Salmonella bacteria: one was modified with digoxin, and the other was modified with biotin. In the presence of the biotin- and digoxin-modified primers and target DNA, the RPA produced a substantial amount of duplex DNA attached to biotin and digoxin. The products were detected using LF strips through immunoreaction: anti-digoxin antibodies on the gold nanoparticles, digoxin on the duplex, streptavidin on the LF test line, and biotin on the duplex. The developed RPA-LF assay allowed detection of Salmonella genomic DNA in less than 20 min with simple water bath equipment or portable thermal equipment. In addition, the RPA-LF assay was highly sensitive, with a detection limit as low as 20 fg of target DNA or 1.05 × 10¹ cfu of bacteria in pure culture, and highly specific, exhibiting no cross-reaction with Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Enterobacter aerogenes, or Campylobacter jejuni. Importantly, Salmonella could be detected in milk and chicken breast at concentrations as low as 1.05 × 10⁰ cfu/mL or 1.05 × 10⁰ cfu/g after enrichment for 2 h and in eggs at 1.05 × 10⁰ cfu/g after enrichment for 4 h. Furthermore, RPA was more sensitive than PCR, which requires a thermal cycling device. In summary, this study describes a sensitive, simple, and point-of-use detection method for Salmonella bacteria.