A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products

The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen. The rapid and sensitive detection of L. monocytogenes is important in the food industry as well as in medical diagnosis. In this study, a PCR-based method for the rapid, specific, and sensitive detection of L. monocytogenes in food products was developed. The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L. monocytogenes within the Listeria genus. The primers for the Listeria genus and for L. monocytogenes were used in the same reaction mix for their simultaneous detection. In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control. For the detection of Listeria and L. monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction. The method detects 1 to 5 CFU in a 25-g sample in < or = 24 h. It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.     

Rapid enumeration of Listeria monocytogenes in milk using competitive PCR

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in milk. Sterile milk was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate/phenol/chloroform, followed by PCR. Several primers for L. monocytogenes hlyA gene were tested for specific detection and DG69/DG74 primer set was selected. The primer set produced a 636-bp band from L. monocytogenes, but no band appeared from the other six Listeria spp. tested. A detection limit was as few as 10(3) colony-forming unit (cfu) per 0.5 ml of milk with this primer set. When the samples were cultured at 25 degrees C for 15 h in a TSBY medium, even a single bacterium could be detected with this primer set by PCR. For the cPCR, hlyA gene segment was cloned in pGem-4Z vector and was modified to produce competitor DNA. The competitor DNA has the same primer binding sites and sequences as the target DNA except EcoRI site. Known amount of competitor DNA was coamplified with L. monocytogenes total DNA isolated from artificially inoculated milk. The target DNA and competitor DNA were distinguished by EcoRI digestion after cPCR. The cell number determined by cPCR was approximately equal to the colony-forming unit from conventional plate counting method. For the whole procedure, it took only 5 h.     

Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products

The application of real-time PCR targeting the multicopy 16S rRNA gene and the single copy recA gene was evaluated for the enumeration of bifidobacteria in 29 probiotic products claimed to contain these organisms. Both assays relied on the use of genus-specific primers and the non-specific SYBR Green I chemistry. For both applications, the calibration curve was constructed using the type strain of Bifidobacterium animalis subsp. lactis. Upon correction with a factor corresponding to the 16S rRNA gene copy number, both assays generally produced comparable enumeration results. Only in exceptional cases, differences between both gene targets were found in probiotic products containing low amounts of bifidobacteria in which case the quantification of the multicopy 16S rRNA gene turned out to be more sensitive than the recA-based assay. On the other hand, the use of the latter single copy gene in real-time PCR quantification offers the advantage that no prior knowledge of bacterial content is required when using genus-specific primers, since no correction for multiple gene copies has to be performed. Only 11 of the analysed products (38%), including one dairy based product and ten dried products, contained a minimal Bifidobacterium concentration of 10(6) CFU per ml or g of product. Depending on the application, both assays proved to be rapid and reproducible alternatives for culture-based detection and quantification of bifidobacteria in probiotic products.     

免疫磁分离技术在食源性单增李斯特菌检测中应用的研究进展

食源性单增李斯特菌是李斯特菌属中的唯一能引起人类疾病的病原菌,致死率30%~70%,并严重威胁着人类健康。早期快速准确地检测出食品中可能污染的单增李斯特菌对于减少死亡率非常重要,因此亟需建立一些快速、灵敏和高特异性的检测方法。现有单增李斯特菌的检测方法对未经前增菌的食品样本检测灵敏度较低,限制了这些方法直接用于食品样本中单增李斯特菌的快速检测。免疫磁分离是一种可以短时间内高效富集样本中目的菌的技术,与常用的检测方法结合,可以缩短检测周期,提高检测灵敏度。本文综述了免疫磁分离技术在食源性单核细胞增生性李斯特检测中应用的研究进展。      

免疫磁分离技术在食源性单增李斯特菌检测中应用的研究进展

食源性单增李斯特菌是李斯特菌属中的唯一能引起人类疾病的病原菌,致死率30%~70%,并严重威胁着人类健康。早期快速准确地检测出食品中可能污染的单增李斯特菌对于减少死亡率非常重要,因此亟需建立一些快速、灵敏和高特异性的检测方法。现有单增李斯特菌的检测方法对未经前增菌的食品样本检测灵敏度较低,限制了这些方法直接用于食品样本中单增李斯特菌的快速检测。免疫磁分离是一种可以短时间内高效富集样本中目的菌的技术,与常用的检测方法结合,可以缩短检测周期,提高检测灵敏度。本文综述了免疫磁分离技术在食源性单核细胞增生性李斯特检测中应用的研究进展。     

Comparison of different most-probable-number methods for enumeration of Listeria in poultry

To estimate levels of Listeria spp. in poultry and to select the most appropriate enumeration method for routine analysis, 40 naturally contaminated retail chicken carcasses were tested in Ponferrada (León, N.W. Spain) using the direct plate count technique and various most-probable-number (MPN) designs (UVM I [University of Vermont modified Listeria enrichment broth], Fraser enrichment broth, or both were used in 3-, 5-, and 10-tube MPN techniques). MPN estimation was obtained from the number of tubes with Listeria confirmed (after streaking on PALCAM and modified Oxford agars: "true" MPN) and from the number of dark Fraser broth tubes ("predictive" MPN). Samples were analyzed in duplicate. Low levels of Listeria were found (< 110 CFU/g). The direct plate count technique was totally ineffective for enumerating Listeria in poultry. The single-step (UVM I) and the two-step (UVM I-Fraser) MPN methods gave comparable estimations and a low number of significantly discrepant predictions. Using a single-step method with Fraser broth, lower true MPNs were obtained. The number of tubes used (3, 5, or 10) did not have a substantial influence on the results. Similar estimations, highly correlated (r = 0.538 to 0.968; P < 0.001), were found with (true MPN) and without (predictive MPN) plating confirmation when using the two-step MPN method. The statistical evaluation of the differential character of Fraser broth as part of the two-step MPN method showed high sensitivity (87.5 to 92.5%), specificity (95.2 to 98.6%), efficiency (94.2 to 97.6%), and predictive values (73.6 to 89.9% for a positive test and 98.0 to 98.9% for a negative test). Taking into account these results, we suggest the convenience of using a 3- or 5-tube two-step (UVM I-Fraser) MPN method with estimations obtained from the number of tubes with darkening, without confirmation, in order to achieve great savings in time and money.     

食源性变形杆菌氨基糖苷类修饰酶基因及16S rRNA甲基化酶基因研究

目的了解食源性变形杆菌的氨基糖苷类耐药基因情况。方法收集2011—2014年石家庄市售各类食品中分离到的对阿米卡星和庆大霉素至少有一种耐药的变形杆菌124株,用PCR、测序和基因库在线比对方法分析6种氨基糖苷类修饰酶基因与3种16S r RNA甲基化酶基因。结果 124株变形杆菌中氨基糖苷类修饰酶耐药基因aac C2、aac A4、aad A1和aph A6的检出率分别为95.2%(118/124)、80.6%(100/124)、73.4%(91/124)和5.6%(7/124);16S r RNA甲基化酶耐药基因rmt B检出率为87.1%(108/124)。aac C1、aad B、arm A和rmt C基因均未检出。结论 aac C2型氨基糖苷类修饰酶基因与rmt B型16S r RNA甲基化酶基因流行是食源性变形杆菌对氨基糖苷类抗生素耐药的重要原因。     

食品中单核增生性李斯特菌的PCR快速检测研究

为提高食品中单增李斯特菌的检测水平,针对单增李斯特菌中多个稳定的特异性基因hlyA、plcB、prfA、iap,设计并筛选出7对引物组成多重-巢式PCR联合检测体系,并结合高灵敏性的聚丙烯酰胺凝胶电泳,对单增李斯特菌进行快速检测,多重PCR的灵敏度达到1×102CFU ml,巢式PCR的灵敏度达到1×10CFU ml。结果表明该检测体系具有快速可靠、灵敏准确及特异性好的特点,而且有效缩短了检验周期,从传统的7～14d缩短到1～2d。     

A multiplex PCR assay based on 16S rRNA and hly for rapid detection of L. monocytogenes in Milk

A multiplex PCR assay was developed by targeting ‘16S rRNA’ and ‘hly’ genes for detection of Listeria or Listeria monocytogenes in dairy foods on the basis of amplification of 1200 and 713 bp products, respectively. The assay conditions were optimized to make it truly rapid and to cut down the cost. The authenticity of the multiplex PCR was ascertained by using Nested PCR targeted against internal region of ‘hly’ gene that produced an amplified product of 188 bp. The multiplex PCR assay was found to be specific for detection of L. monocytogenes only since none of the non-listerial cultures gave positive signal. The sensitivity of the multiplex PCR was limited to 10 ng pure DNA and 1–10 cells of L. monocytogenes after 4–6 h enrichment in Listeria enrichment broth. When applied to 20 raw milk and 10 pasteurized milk samples, L. monocytogenes could not be detected in any of the samples by the multiplex PCR assay. This assay could find potential application in dairy industry for monitoring dairy foods for this high risk food pathogen on routine basis.     

Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes

A multiplex SYBR Green I-based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein (fimI) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene (hly) of L. monocytogenes. These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75 degrees C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green-based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes, respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1 microl of culture to enable direct detection with the LightCycler.     

FTA滤膜用于PCR检测单核细胞增生李斯特氏菌的研究

建立单核细胞增生李斯特氏菌(Listeria moncytones,LM)快速、敏感、特异的PCR检测方法.利用FTA滤膜提取模板DNA,采用PCR特异性扩增单增李斯特菌的溶血素基因(HIyA),并评价该方法的特异性与灵敏性.引物能特异性的扩增单增李斯特的HIyA基因,而其他细菌的扩增结果均呈现阴性:利用FTA滤膜提取模板直接检测单增李斯特具有较高的灵敏度,灵敏度为l 02 cfu/mL.利用FFA滤膜提取模板,操作简便,成本低且具有较高的灵敏度,为食品中单核细胞增生李斯特氏菌的快速检测提供新的手段.     

基于双启动引物特异性检测单核细胞增生李斯特氏菌的PCR方法

以单核细胞增生李斯特氏菌iap基因为靶基因,利用一新型PCR引物设计方法--双启动引物（Dual-priming oligonucleotide,DPO）,建立了特异性检测单核细胞增生李斯特氏菌的DPO-PCR方法,测试了DPO-PCR方法退火温度不敏感性、特异性及灵敏度,并在实践检测中进行了初步应用。结果显示:该方法检测单核细胞增生李斯特氏菌的灵敏度为1.51×102CFU/mL;退火温度不敏感性测试中,与常规PCR引物相比,DPO引物在48⁶8℃退火温度范围内均能够高效率地扩增靶基因;特异性测试中,DPO-PCR方法能特异地检测出目标菌,与其他菌株无非特异性扩增反应,比常规PCR方法显示出更强的特异性。实践应用证明,利用DPO-PCR方法对130份样本进行检测,共计检出9份单核细胞增生李斯特氏菌阳性样本,经国标法（GB/T 4789.30-2008）复检,两者检测结果一致,显示出良好的实用性,为单核细胞增生李斯特氏菌的快速准确检测提供了新方法。      

实时定量荧光PCR快速鉴定食品中单核细胞增生李斯特氏菌

目的建立实时定量荧光PCR法(real-time PCR)快速鉴定食品中的单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)。方法选取2016年国家食品风险监测样本134例与模拟灭活LM样本10例,采用GB/T4789.30-2010与real-time PCR方法同步检测单核细胞增生李斯特菌。结果共检测食品134份,包括肉制品、水产品、快餐和即食食品等。共检出8株LM,检出率为5.97%。以GB/T4789.30-2010为金标准判断,real-time PCR方法检测样本中LM的灵敏度与特异度均达到100%。模拟灭活LM样本real-time PCR方法检出率为100%,标准法检出率为0%。结论本方法可以简化实验程序,减少工作量,节约检测试剂,为可能发生的食物中毒尽早提供实验依据。     

实时荧光PCR定量检测食品中单增李斯特菌

目的建立快速、敏感、特异的食品中单增李斯特菌检测方法。方法针对单增李斯特菌溶素A基因（hlyA）设计一对引物和一条探针，并用该引物和探针运用实时荧光PCR技术对单增李斯特菌的DNA、细胞、质粒和样品进行实时荧光PCR定量检测。结果利用实时荧光PCR技术，建立了DNA校正曲线、细胞校正曲线和质粒校正曲线。DNA校正曲线在1～32CFU/ml、细胞校正曲线在32—320CFU／ml、质粒校正曲线在1—37Copies／ml，线形关系良好，且三种校正曲线检测样品得出的结果基本吻合。结论本试验建立起来的实时荧光PCR定量检测单增李斯特菌的方法灵敏度高、特异性好、准确，可应用于食品中单增李斯特菌的检测。     

An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica

A molecular method for the detection of Salmonella enterica strains based on 16S rRNA sequence analysis was developed by a modification of the previously described PCR primer 16SFI [J. Appl. Bacteriol. 80 (1996) 659], which was combined with a newly developed primer annealing at the position 66-82. Only approximately two thirds of now determined Salmonella 16S rRNA sequences contained a region identical to the 16SFI primer sequence and the reverse primer 16SIII was also not specific. Combined, these two primers have been claimed to allow the specific detection of all Salmonella; however, in this study, they did not recognize S. bongori and 3 out of 78 tested S. enterica strains. They also identified some of the tested Enterobacter cloacae strains as Salmonella. On the contrary, the new primer pair, MINf and MINr, made it possible to recognize correctly all of the 78 tested S. enterica strains, representing 31 different Salmonella serovars. None of the 23 non-Salmonella strains from the related gamma-proteobacterial genera was incorrectly recognized as belonging to S. enterica.     

Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR

The objective of this study was to develop a method combining nanoparticle-based immunomagnetic separation (IMS) with real-time PCR for a rapid and quantitative detection of Listeria monocytogenes. Carboxyl modified magnetic nanoparticles were covalently bound with rabbit anti-L. monocytogenes via the amine groups. Several factors, such as the amount of immunomagnetic nanoparticles (IMNPs), reaction and collection times, and washing step, were optimized, and the nanoparticle-based IMS in combination with real-time PCR was further evaluated for detecting L. monocytogenes from artificially contaminated milk. The cell numbers calculated from the means of threshold cycles (CT) of PCR amplification curves were compared to those from plate counts in order to determine the correspondence degree of quantitative data. The capture efficiency (CE) by plating from IMNP-based IMS was 1.4 to 26 times higher than those of Dynabeads-based IMS depending on the initial cell concentrations inoculated into milk samples. When combined with real-time PCR, L. monocytogenes DNA was detected in milk samples with L. monocytogenes >or=10(2) CFU/0.5 ml. In the range of 10(3) to 10(7)L. monocytogenes CFU/0.5 ml, cell numbers calculated from CT values were 1.5 to 7 times higher than those derived from plate counts. Our results demonstrated that both the use of nanoparticles and the choice of anti-L. monocytogenes in our IMNP-based IMS in combination with real-time PCR has improved the sensitivity of L. monocytogenes detection from both nutrient broth and milk samples.     

单核细胞增生李斯特氏菌的PCR检测方法

研究比较了裂解法、热煮沸法、试验盒法提取单增李斯特氏菌DNA的效果 ,认为Promega试剂盒法提取的DNA ,得率高、纯度好。同时根据单增李斯特氏菌的毒力相关基因的 5对引物 ,进行了引物的特异性研究 ,结果发现inlA引物、inlB引物、plcA引物特异性好 ,可以用于食品中单增李斯特氏菌的检测     

苏州市食源性单核细胞增生李斯特菌的全基因组测序分析

目的 应用高通量测序技术分析苏州市食源性单核细胞增生李斯特菌(Listeria monocytogenes, Lm)的毒力基因携带情况、分子分型、遗传进化谱系及遗传进化关系等分子特征。方法 对2016—2020年分离自食品的42株Lm进行全基因组测序,运用CLC Genomics Workbench 21.0.4软件进行组装及毒力基因和耐药基因分析;通过与BIGSdb-Lm数据库比对获得谱系、克隆群(clone complexes,CC)、血清群、多位点序列分型(multilocus sequence typing,MLST)、核心基因组多位点序列分型(core genome multilocus sequence typing,cgMLST);利用柏熠微生物分析平台v4.0构建最大似然树。结果 42株Lm共包含两个谱系,谱系Ⅰ和谱系Ⅱ,以谱系Ⅱ为主(83.3%)。血清群分为Ⅱ_a、Ⅱ_b和Ⅱ_c,以Ⅱ_a为主(57.1%)。42株Lm分为11个CC型, 11个序列型(sequence type, ST型)...     

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein,rimM, for detection and enumeration of Streptococcus thermophilus in dairy products

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein, rimM, for specific detection of Streptococcus thermophilus was developed. The designed real-time PCR primers and probe were specific for S. thermophilus JCM20026, LMG6896, LMG18311, OJT101, OJT102 but not Enteroccocus spp., Lactococcus lactis subsp. lactis, and Streptococcus salivarius which are phylogenetically closely related to S. thermophilus and are difficult to identify using culture-based methods. The linear range of the developed real-time PCR method was from 2.7 to 8.6 log CFU ml^?1 with an amplification efficiency of 96%. Minor differences (about 0.4 log CFU ml^?1) were observed between counts of S. thermophilus obtained by culture and real-time PCR method in plain yoghurt and yoghurt containing fruits. Therefore, the developed real-time PCR method could be of potential application in specific detection and accurate enumeration of S. thermophilus in a wide range of dairy products.     

Simultaneous detection of Listeria monocytogenes and Salmonella by multiplex PCR in cooked ham

A multiplex PCR (m-PCR) assay with an internal amplification control (IAC) was developed for the simultaneous detection of Salmonella spp. and Listeria monocytogenes through invA and prfA genes, respectively. To ensure the detection of the pathogens in cooked ham, samples were enriched in both buffered peptone-water and Half Fraser broth. Subsequently, equal volumes of enrichment broths were mixed and DNA purification was performed prior to m-PCR reaction, saving considerable time and effort. The m-PCR also proved to be very useful as a simple and ready-to-go method for simultaneous confirmation of presumptive L. monocytogenes and Salmonella spp. colonies directly from agar plates without any DNA extraction steps.