单核细胞增多性李斯特菌PCR和PCR-ELISA检测方法的建立
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摘要
李斯特菌属(Listeria spp.)是一类革兰氏阳性菌,属内包括单核细胞增多性李斯特菌(L.monocytogenes)、绵羊李斯特菌(L.ivanovii)、英诺克李斯特菌(L.innocua)、威尔斯李斯特菌(L.welshimeri)、西尔李斯特菌(L.seeligeri)和格氏李斯特菌(L.grayi.)等6个种。李斯特菌在自然界中广泛存在,其中单核细胞增多性李斯特菌(Listeria monocytogenes,LM)对人和多种动物具有致病性,在国内外食品安全检测与控制中日益受到重视。LM在4℃的环境中仍可生长繁殖,是冷藏食品中威胁人类健康的主要病原菌之一。同时,单核细胞增多李斯特菌作为研究胞内菌感染和细胞免疫的良好模型,已经引起越来越多研究者的关注。目前,国内对LM的研究尚不深入,工作实践中使用的检验方法也主要采用二次增菌后分离鉴定,烦琐、费时,迫切需要建立敏感、特异、高通量的分子生物学检测方法。在LM中存在两个主要的毒力岛,即LIPI-1毒力岛(Listeria pathogenicity island 1)和LIPI-2(Listeria pathogenicityisland 2),其中,IPIL-1也称为prfA基因簇,是主要毒力因素,由prfA、pica、hly、mpl、actA和plcB构成,由prfA进行表达调控。本研究旨在对食品中单核细胞增多性李斯特菌污染状况、LM的生物学特性、LIPI-1毒力岛全基因序列以及分子生物学检测方法进行研究,为系统开展LM致病机理研究、开发新型疫苗载体等工作奠定基础,为检验检疫工作提供检测技术支持,为开展食品中LM的防控提供依据。
1.单核细胞增多性李斯特菌的分离鉴定与生物学特性研究
按照检验检疫行业标准(SN/T0148.1-2005)对156个动物源性食品样品进行了LM的分离鉴定,从中分离到7株单核细胞增多性李斯特菌,表明在动物源性食品中存在LM污染,该结果将为进行食品中LM的风险评估等工作提供参考依据。以参考菌株CCTCCAB97021和C53004为参照,对LM分离菌株XFL0605进行了药敏试验、小鼠致病性试验(LD_(50)测定)和溶血活性检测。在溶血素(LLO)溶血活性实验中,相同培养条件下,3个试验菌株培养上清中LLO的含量存在差异,但产生的单位浓度LLO活性相当,推测菌株XFL0605产生LLO的能力较参考菌株CCTCCAB97021和C53004 2株LM菌株弱。分离菌株XFL0605培养上清溶血活性与其它试验菌株相当,但小鼠毒力(LD_(50))方面较其它实验菌株略低,该菌对昆明鼠的LD_(50)为2.58×10~5CFU/mL,推测该菌株致病性差异可能由于LLO等相关毒力因子的表达差异所致。试验结果还证明,溶血素在pH5.6时活性最高,在pH7.0时活性基本丧失。对实验菌株的药敏实验结果表明,分离菌株XFL0605对常用药物(氨苄青霉素、链霉素、氯霉素、卡那霉素、环丙沙星和诺氟沙星)敏感性较高,为在分子生物学改造研究中,选择抗性筛选标记提供了依据。
为方便、精确的检测溶血素活性,建立了分光光度法检测LM溶血素(LLO)活性,本方法中绵羊红细胞(SRBC)浓度与A_(540)相关系数为0.99995,表现出明显的线性关系,能够直接对LLO溶血活性进行量化分析。分离菌株XFL0605、2个参考菌株C53004和CCTCCAB97021培养上清半数溶血值(HC_(50))测定结果分别是18.85、19.68和21.38,与测定的LLO含量(分别为1.305、1.530和1.749mg/mL)的呈正相关性,表明3个LM菌株产生的单位含量LLO溶血活性相当,该结论在对3个菌株hlyA基因克隆测序后得到印证。本研究建立的分光光度法检测LM溶血素活性的方法具有相关系数高、敏感和判定客观的特点,较好的解决了菌株LLO溶血活性检测与鉴定问题,为进一步测定单核细胞增多性李斯特菌溶血素(LLO)编码基因表达产物活性检测奠定基础。同时,本研究还改进和建立了微量反应板梯度稀释法检测溶血素的方法,该方法直观、操作方便。
2.单核细胞增多性李斯特菌分子生物学检测方法研究
针对LM的IdyA基因设计特异性引物和探针,PCR扩增目的片段长度为348bp,建立了PCR和PCR-ELISA检测食品样品中单核细胞增多性李斯特菌的方法。本研究建立的PCR-ELISA方法中,在上游引物5'端标记生物素,核酸探针的3'端标记地高辛,将PCR扩增、核酸液相杂交以及酶联显色技术结合,实现扩增产物上标记的生物素与包被在微孔板上的链霉亲和素结合,标记有地高辛的探针与产物杂交,再与碱性磷酸酶标记的抗地高辛抗体结合,经底物显色。经对试验菌株检测表明,PCR、PCR-ELISA方法检测单核细胞增多性李斯特菌能扩增出特异片段,其它李斯特属的绵羊李斯特菌、英诺克李斯特菌、威尔斯李斯特菌、西尔李斯特菌和格氏李斯特菌、大肠杆菌、葡萄球菌和沙门氏菌等扩增检测结果是阴性。对相关条件进行了优化,当NaOH浓度为0.3 mol/L,杂交时间60 min;酶反应时间为45 min时,结果较好,洗板液以pH7.4 TBS-T为最佳。与PCR产物电泳法检测比较,PCR-ELISA的敏感性是前者156倍以上,并适合批量样品的检测分析。对60个样品检测表明,与SN/T0148.1-2005方法(引用ISO11290-1:2004)相比,PCR和PCR-ELISA方法具有更高的敏感性,表明本方法可以用于肉类食品中单核细胞增多性李斯特菌的快速诊断,具有敏感、特异、快速,重复性良好,无溴乙锭污染以及适合批量检测等优点,进一步标准化后可望作为食品中单核细胞增多性李斯特菌的常规检测方法。
3.单核细胞增多性李斯特菌IPIL-1全基因克隆与序列分析
参考Genbank中的单核细胞增多性李斯特菌LIPI-1有关基因序列设计引物,分段扩增,再进行编码核苷酸序列拼接,克隆了单核细胞增多性李斯特菌分离菌株XFL0605 LIPI-1毒力岛全基因,序列全长为8558bp,Accession No.EF661572,包括了完整的prfA、plcA、hly、mpl、actA和plcB等6个基因。
对单核细胞增多性李斯特菌XFL0605株LIPI-1编码的6个毒力基因核苷酸序列进行分析表明,各毒力基因反映出来的进化关系不尽一致,表明该菌株在获得LIPI-1外源性毒力基因方面具有不同步性,各毒力基因的来源也不尽相同。对XFL0605菌株中LIPI-1各毒力基因所推导氨基酸序列N端作信号肽预测分析结果表明,PlcA、LLO、Mpl、ActA和PlcB5个毒力蛋白中均存在信号肽序列;actA基因推导氨基酸序列中存在跨膜区(578-600位编码氨基酸),其跨膜蛋白为Ⅰ型。LIPI-1序列中存在5个14bp的PrfA蛋白结合区,分别位于plcA上游59-73bp处,prfA上游62-75bp处,hlyA基因的上游183-196bp处,mpl上游185-198bp和actA基因上游185-198bp处。这些核苷酸序列是调控蛋白PrfA的结合位点,并具有回文结构的特点。
对3个菌株CXFL0605、C53004和CCTCCAB97021)hlyA基因和推导氨基酸序列进行了分析,发现三者具有高度同源性,hly基因同源性在96.9%——100%之间,氨基酸同源性在97.9%以上,并具有相同的PEST基序和C端保守11肽序列,表明所测定3个菌株培养上清中LLO活性基本相当具有遗传基础。对克隆的hlyA基因所推导溶血素氨基酸序列进行PEST分析结果表明,编码LLO的N端32-50位氨基酸序列(KENSISSMAPPASPPASPK)为富含P(脯氨酸)、E(谷氨酸)、S(丝氨酸)和T(苏氨酸)的PEST(Pro-Glu-Ser-Thr)基序,PEST find值为4.71。试验菌株XFL0605actA基因编码604个氨基酸,缺失了105bp富脯氨酸重复片段编码碱基,只有两个E/DFPPPPXD/E重复序列。本研究中发现3株单核细胞增多性李斯特菌溶血素中PEST基序的氨基酸序列完全相同,而且存在PPAS氨基酸重复序列。3个菌株LLO氨基酸序列的C端483-493位存在相同的保守11个氨基酸多肽(序列为ECTGLAWEWWR),该11肽保守序列是CDTX类毒素的特征性序列。
The genus Listeria is a group of Gram-positive bacteria widely dispersed in the environment and foods,and currently subdivided into six species,L.monocytogenes,L. seeligeri,L.welshimeri,L.innocua,L.ivanovii,and L.grayi.Only L.monocytogenes is a human pathogen and a food-borne pathogen that can cause Listerisis with a high mortality rate.Listeriosis is a clinically serious disease with fatality rate of about 20 percents.Its ability to grow at temperatures as low as 4℃permits multiplication in refrigerated foods. In recent years,L.monocytogenes has become not only an important paradigm for immunological investigation but also an important model system for analysis of the molecular mechanisms of intracellular parasitism.The present method for analysis L. monocytogenes is complex and time consuming,requires 24 and 48 hours of enrichment, followed by a variety of other tests.It is necessary to establish a simple molecular method fits for rapid screen and confirmation of suspected isolates.Listeria monocytogenes strain contain two virulence clusters,Listeria pathogenicity island 1(LIPI-1) and Internalin Island(LIPI-2).LIPI-1 is the Central Virulencee Gene Cluster encode prfA,plcA,hly, mpl,actA,and plcB.In this research,we clone,Sequence,and analysis of the LIPI-1 gene duster from a L.monocytogenes isolate strain XFL0605.Furthermore,a simple PCR and PCR-ELISA method for LM detection was developed.
The main research included as below.
1.Isolation and biological characterization of Listeria monocytogenes Seven strains of L.monocytogenes were isolated from 156 food samples follows standard method SN/T0148.1-2005.Virulence determination,haemolytic activity detemination, and antibiotic sensitivity test were explored to the isolate L.monocytogenes strain XFL0605 and reference strains CCTCCAB97021 and SC53004.A simple and sensitive ultraviolet spectrophotometry method was established for determining the haemolytic activity of LM strains.The coefficient between SRBC condensity and A540 was 0.99995. The HC_(50) values of XFL0605、CCTCCAB97021 and C53004 culture supematants were 18.85,21.38,and19.68 respectively,showing a strong eorelationship to the set of LLO concentrations.It showed that the haemolytie activity of the LLO secreted from the 3 different strains was identical which was confirmed by sequences of hly gene encoding the pore-forming cytolysin listeriolysin.While XFL0605 strain,whose LD_(50) was 2.58×10~5,was less pathogenic to mice compare to the reference strains.Close relationship between haemolytic activity in the culture supematants and the pH values was found, LLOs were active at pH value of 5.6 while inactive at pH value of 7.0.Antibiotic sensitivity test showed that 3 LM strains were susceptibility to 7 kinds of antimicrobial agents including Ampicillin,Streptomycin,Chloromycetin,Kanamycin,Tetracycline, Norfloxacin and Ciprofloxacin.The spectrophotometric method developed in this study for LLO haemolytic activity testing was direct-viewing and sensitive,and is suitable for testing the expression of hly gene.At the same time,the microtitre plate with serial dilution method was developed and modified which was simple and convenient for LLO haemolytic activity testing.A microtitre plate testing method measure LLO haemolytic ative by serial dilution was developed.
2.Development of molecular methods for Listeria monocytogenes detection
To detect L.monocytogenes contamination in foods,the simple PCR and PCR-ELISA method were developed,according to the published hlyA gene sequence,a forward PCR primers labled with biotin at 5" end and a specific probe labled with digxin were designed by using primer 5.0 software.The PCR-ELISA method which was developed in this study combinated PCR,nucleic acid hybrid reaction and enzyme coloration.Only L. monocytogenes strains were successfully amplified the expected fragment,not from E.coli,Salmonella,L.iuanuii,L.innocua,L.innocua,L.seeligeri,and L.gray were negative. The method was then applied to detect L.monocytogenes in 60 food samples to validate the PCR-ELISA procedures by compareing to the isolate method,which was higher positive detection than SN/T0148.1-2005(devivative from ISO11290-1:2004 ).LM detection can be improved by PCR-ELISA the sensitivity of PCR products 156-folds higher than agarose gel electrophoresis.The system allowed quantitative results to be obtained within 5h after 16h of enrichment in half-Fraser broth and was relatively cost-effective,showing a good potential for routine analytical use.
3.Cloning and phylogenetic analysis of LIPI-1 gene cluster from XFL0605 strain
The LIPI-1 gene cluster of Listeria monocytogenes XFL0605 strain was segmentally amplified by PCR and analyses.The result showed that LIPI-1 of i XFL0605 strain was 8558nt(Genbank accesion No.EF661572),contained all the members of the prfA-regulated virulence gene cluster prfA,hly,pleA,plcB,mpl,and actA.Phylogenetic analysis XFL0605 strain and other L.monocytogenes,L.seeligeri and L.ivanovii strains on the base of sequence of the LIPI-1 and prfA,hly,plcA,plcB,mpl,and actA gene individal clearly demonstrated that the evolutionary relationship of strains were difference. It revealed that recombination of LIPI-1 in different assembly process and from different source.Furthermore,the signal peptides and transmember domains and their cleavage positions were prodicted,and investigated the PrfA DNA binding sites of the LIPI-1 virulence genes.
After prediction and analysis of signal peptides of the LIPI-1 of XFL0605 strain by the bio-software SignalP3.0,proteins of PlcA,LLO,Mpl,ActA and PlcB have an average of 26.6 amino acids located at the N-terminal region.Only the membrane protein which was essential for bacterial actin-based motility encoded by actA has the transmember domain(578-600 amino acids) in C-terminus region.There were five 14-bp DNA palindromic sequence(PrfA DNA bingding site sequence) that were present in the target gene promoters of prfA,picA,hlyA,actA and actA of XFL0605.All these palindrome domains were the targets for PrfA recognizes and binding.
Analysis of hlyA gene and deduced amino acid of the three L.monocytogenes(XFL0605, C53004 and CCTCCAB97021) revealed that the nucleotide homology ranging from 96.9%to 100%,and deduced amino acid sequence homology of over 97.9%.And the three LLOs contained a same 19-amino acid PEST-like sequence (KENSISSMAPPASPPASPK) at the N-terminus region which is essential for the virulence and intracellular compartmentalization of LM,and a same conserved undecapeptide,ECTGLAWEWWR,at the C terminus region which is the characterization of the CDTX toxin.The results supported the conclusion of the previous study of hemolytic activity of the three LM strains.ActA gene of XFL0605 encodes 604 amino acids and contains whole open reading frame but 105bp nucleotides were absent from in proline-rich region,only contains two E/DFPPPPXD/E repeat sequences.We sugest the variation contributes to virulence difference in mice.
1.单核细胞增多性李斯特菌的分离鉴定与生物学特性研究
按照检验检疫行业标准(SN/T0148.1-2005)对156个动物源性食品样品进行了LM的分离鉴定,从中分离到7株单核细胞增多性李斯特菌,表明在动物源性食品中存在LM污染,该结果将为进行食品中LM的风险评估等工作提供参考依据。以参考菌株CCTCCAB97021和C53004为参照,对LM分离菌株XFL0605进行了药敏试验、小鼠致病性试验(LD_(50)测定)和溶血活性检测。在溶血素(LLO)溶血活性实验中,相同培养条件下,3个试验菌株培养上清中LLO的含量存在差异,但产生的单位浓度LLO活性相当,推测菌株XFL0605产生LLO的能力较参考菌株CCTCCAB97021和C53004 2株LM菌株弱。分离菌株XFL0605培养上清溶血活性与其它试验菌株相当,但小鼠毒力(LD_(50))方面较其它实验菌株略低,该菌对昆明鼠的LD_(50)为2.58×10~5CFU/mL,推测该菌株致病性差异可能由于LLO等相关毒力因子的表达差异所致。试验结果还证明,溶血素在pH5.6时活性最高,在pH7.0时活性基本丧失。对实验菌株的药敏实验结果表明,分离菌株XFL0605对常用药物(氨苄青霉素、链霉素、氯霉素、卡那霉素、环丙沙星和诺氟沙星)敏感性较高,为在分子生物学改造研究中,选择抗性筛选标记提供了依据。
为方便、精确的检测溶血素活性,建立了分光光度法检测LM溶血素(LLO)活性,本方法中绵羊红细胞(SRBC)浓度与A_(540)相关系数为0.99995,表现出明显的线性关系,能够直接对LLO溶血活性进行量化分析。分离菌株XFL0605、2个参考菌株C53004和CCTCCAB97021培养上清半数溶血值(HC_(50))测定结果分别是18.85、19.68和21.38,与测定的LLO含量(分别为1.305、1.530和1.749mg/mL)的呈正相关性,表明3个LM菌株产生的单位含量LLO溶血活性相当,该结论在对3个菌株hlyA基因克隆测序后得到印证。本研究建立的分光光度法检测LM溶血素活性的方法具有相关系数高、敏感和判定客观的特点,较好的解决了菌株LLO溶血活性检测与鉴定问题,为进一步测定单核细胞增多性李斯特菌溶血素(LLO)编码基因表达产物活性检测奠定基础。同时,本研究还改进和建立了微量反应板梯度稀释法检测溶血素的方法,该方法直观、操作方便。
2.单核细胞增多性李斯特菌分子生物学检测方法研究
针对LM的IdyA基因设计特异性引物和探针,PCR扩增目的片段长度为348bp,建立了PCR和PCR-ELISA检测食品样品中单核细胞增多性李斯特菌的方法。本研究建立的PCR-ELISA方法中,在上游引物5'端标记生物素,核酸探针的3'端标记地高辛,将PCR扩增、核酸液相杂交以及酶联显色技术结合,实现扩增产物上标记的生物素与包被在微孔板上的链霉亲和素结合,标记有地高辛的探针与产物杂交,再与碱性磷酸酶标记的抗地高辛抗体结合,经底物显色。经对试验菌株检测表明,PCR、PCR-ELISA方法检测单核细胞增多性李斯特菌能扩增出特异片段,其它李斯特属的绵羊李斯特菌、英诺克李斯特菌、威尔斯李斯特菌、西尔李斯特菌和格氏李斯特菌、大肠杆菌、葡萄球菌和沙门氏菌等扩增检测结果是阴性。对相关条件进行了优化,当NaOH浓度为0.3 mol/L,杂交时间60 min;酶反应时间为45 min时,结果较好,洗板液以pH7.4 TBS-T为最佳。与PCR产物电泳法检测比较,PCR-ELISA的敏感性是前者156倍以上,并适合批量样品的检测分析。对60个样品检测表明,与SN/T0148.1-2005方法(引用ISO11290-1:2004)相比,PCR和PCR-ELISA方法具有更高的敏感性,表明本方法可以用于肉类食品中单核细胞增多性李斯特菌的快速诊断,具有敏感、特异、快速,重复性良好,无溴乙锭污染以及适合批量检测等优点,进一步标准化后可望作为食品中单核细胞增多性李斯特菌的常规检测方法。
3.单核细胞增多性李斯特菌IPIL-1全基因克隆与序列分析
参考Genbank中的单核细胞增多性李斯特菌LIPI-1有关基因序列设计引物,分段扩增,再进行编码核苷酸序列拼接,克隆了单核细胞增多性李斯特菌分离菌株XFL0605 LIPI-1毒力岛全基因,序列全长为8558bp,Accession No.EF661572,包括了完整的prfA、plcA、hly、mpl、actA和plcB等6个基因。
对单核细胞增多性李斯特菌XFL0605株LIPI-1编码的6个毒力基因核苷酸序列进行分析表明,各毒力基因反映出来的进化关系不尽一致,表明该菌株在获得LIPI-1外源性毒力基因方面具有不同步性,各毒力基因的来源也不尽相同。对XFL0605菌株中LIPI-1各毒力基因所推导氨基酸序列N端作信号肽预测分析结果表明,PlcA、LLO、Mpl、ActA和PlcB5个毒力蛋白中均存在信号肽序列;actA基因推导氨基酸序列中存在跨膜区(578-600位编码氨基酸),其跨膜蛋白为Ⅰ型。LIPI-1序列中存在5个14bp的PrfA蛋白结合区,分别位于plcA上游59-73bp处,prfA上游62-75bp处,hlyA基因的上游183-196bp处,mpl上游185-198bp和actA基因上游185-198bp处。这些核苷酸序列是调控蛋白PrfA的结合位点,并具有回文结构的特点。
对3个菌株CXFL0605、C53004和CCTCCAB97021)hlyA基因和推导氨基酸序列进行了分析,发现三者具有高度同源性,hly基因同源性在96.9%——100%之间,氨基酸同源性在97.9%以上,并具有相同的PEST基序和C端保守11肽序列,表明所测定3个菌株培养上清中LLO活性基本相当具有遗传基础。对克隆的hlyA基因所推导溶血素氨基酸序列进行PEST分析结果表明,编码LLO的N端32-50位氨基酸序列(KENSISSMAPPASPPASPK)为富含P(脯氨酸)、E(谷氨酸)、S(丝氨酸)和T(苏氨酸)的PEST(Pro-Glu-Ser-Thr)基序,PEST find值为4.71。试验菌株XFL0605actA基因编码604个氨基酸,缺失了105bp富脯氨酸重复片段编码碱基,只有两个E/DFPPPPXD/E重复序列。本研究中发现3株单核细胞增多性李斯特菌溶血素中PEST基序的氨基酸序列完全相同,而且存在PPAS氨基酸重复序列。3个菌株LLO氨基酸序列的C端483-493位存在相同的保守11个氨基酸多肽(序列为ECTGLAWEWWR),该11肽保守序列是CDTX类毒素的特征性序列。
The genus Listeria is a group of Gram-positive bacteria widely dispersed in the environment and foods,and currently subdivided into six species,L.monocytogenes,L. seeligeri,L.welshimeri,L.innocua,L.ivanovii,and L.grayi.Only L.monocytogenes is a human pathogen and a food-borne pathogen that can cause Listerisis with a high mortality rate.Listeriosis is a clinically serious disease with fatality rate of about 20 percents.Its ability to grow at temperatures as low as 4℃permits multiplication in refrigerated foods. In recent years,L.monocytogenes has become not only an important paradigm for immunological investigation but also an important model system for analysis of the molecular mechanisms of intracellular parasitism.The present method for analysis L. monocytogenes is complex and time consuming,requires 24 and 48 hours of enrichment, followed by a variety of other tests.It is necessary to establish a simple molecular method fits for rapid screen and confirmation of suspected isolates.Listeria monocytogenes strain contain two virulence clusters,Listeria pathogenicity island 1(LIPI-1) and Internalin Island(LIPI-2).LIPI-1 is the Central Virulencee Gene Cluster encode prfA,plcA,hly, mpl,actA,and plcB.In this research,we clone,Sequence,and analysis of the LIPI-1 gene duster from a L.monocytogenes isolate strain XFL0605.Furthermore,a simple PCR and PCR-ELISA method for LM detection was developed.
The main research included as below.
1.Isolation and biological characterization of Listeria monocytogenes Seven strains of L.monocytogenes were isolated from 156 food samples follows standard method SN/T0148.1-2005.Virulence determination,haemolytic activity detemination, and antibiotic sensitivity test were explored to the isolate L.monocytogenes strain XFL0605 and reference strains CCTCCAB97021 and SC53004.A simple and sensitive ultraviolet spectrophotometry method was established for determining the haemolytic activity of LM strains.The coefficient between SRBC condensity and A540 was 0.99995. The HC_(50) values of XFL0605、CCTCCAB97021 and C53004 culture supematants were 18.85,21.38,and19.68 respectively,showing a strong eorelationship to the set of LLO concentrations.It showed that the haemolytie activity of the LLO secreted from the 3 different strains was identical which was confirmed by sequences of hly gene encoding the pore-forming cytolysin listeriolysin.While XFL0605 strain,whose LD_(50) was 2.58×10~5,was less pathogenic to mice compare to the reference strains.Close relationship between haemolytic activity in the culture supematants and the pH values was found, LLOs were active at pH value of 5.6 while inactive at pH value of 7.0.Antibiotic sensitivity test showed that 3 LM strains were susceptibility to 7 kinds of antimicrobial agents including Ampicillin,Streptomycin,Chloromycetin,Kanamycin,Tetracycline, Norfloxacin and Ciprofloxacin.The spectrophotometric method developed in this study for LLO haemolytic activity testing was direct-viewing and sensitive,and is suitable for testing the expression of hly gene.At the same time,the microtitre plate with serial dilution method was developed and modified which was simple and convenient for LLO haemolytic activity testing.A microtitre plate testing method measure LLO haemolytic ative by serial dilution was developed.
2.Development of molecular methods for Listeria monocytogenes detection
To detect L.monocytogenes contamination in foods,the simple PCR and PCR-ELISA method were developed,according to the published hlyA gene sequence,a forward PCR primers labled with biotin at 5" end and a specific probe labled with digxin were designed by using primer 5.0 software.The PCR-ELISA method which was developed in this study combinated PCR,nucleic acid hybrid reaction and enzyme coloration.Only L. monocytogenes strains were successfully amplified the expected fragment,not from E.coli,Salmonella,L.iuanuii,L.innocua,L.innocua,L.seeligeri,and L.gray were negative. The method was then applied to detect L.monocytogenes in 60 food samples to validate the PCR-ELISA procedures by compareing to the isolate method,which was higher positive detection than SN/T0148.1-2005(devivative from ISO11290-1:2004 ).LM detection can be improved by PCR-ELISA the sensitivity of PCR products 156-folds higher than agarose gel electrophoresis.The system allowed quantitative results to be obtained within 5h after 16h of enrichment in half-Fraser broth and was relatively cost-effective,showing a good potential for routine analytical use.
3.Cloning and phylogenetic analysis of LIPI-1 gene cluster from XFL0605 strain
The LIPI-1 gene cluster of Listeria monocytogenes XFL0605 strain was segmentally amplified by PCR and analyses.The result showed that LIPI-1 of i XFL0605 strain was 8558nt(Genbank accesion No.EF661572),contained all the members of the prfA-regulated virulence gene cluster prfA,hly,pleA,plcB,mpl,and actA.Phylogenetic analysis XFL0605 strain and other L.monocytogenes,L.seeligeri and L.ivanovii strains on the base of sequence of the LIPI-1 and prfA,hly,plcA,plcB,mpl,and actA gene individal clearly demonstrated that the evolutionary relationship of strains were difference. It revealed that recombination of LIPI-1 in different assembly process and from different source.Furthermore,the signal peptides and transmember domains and their cleavage positions were prodicted,and investigated the PrfA DNA binding sites of the LIPI-1 virulence genes.
After prediction and analysis of signal peptides of the LIPI-1 of XFL0605 strain by the bio-software SignalP3.0,proteins of PlcA,LLO,Mpl,ActA and PlcB have an average of 26.6 amino acids located at the N-terminal region.Only the membrane protein which was essential for bacterial actin-based motility encoded by actA has the transmember domain(578-600 amino acids) in C-terminus region.There were five 14-bp DNA palindromic sequence(PrfA DNA bingding site sequence) that were present in the target gene promoters of prfA,picA,hlyA,actA and actA of XFL0605.All these palindrome domains were the targets for PrfA recognizes and binding.
Analysis of hlyA gene and deduced amino acid of the three L.monocytogenes(XFL0605, C53004 and CCTCCAB97021) revealed that the nucleotide homology ranging from 96.9%to 100%,and deduced amino acid sequence homology of over 97.9%.And the three LLOs contained a same 19-amino acid PEST-like sequence (KENSISSMAPPASPPASPK) at the N-terminus region which is essential for the virulence and intracellular compartmentalization of LM,and a same conserved undecapeptide,ECTGLAWEWWR,at the C terminus region which is the characterization of the CDTX toxin.The results supported the conclusion of the previous study of hemolytic activity of the three LM strains.ActA gene of XFL0605 encodes 604 amino acids and contains whole open reading frame but 105bp nucleotides were absent from in proline-rich region,only contains two E/DFPPPPXD/E repeat sequences.We sugest the variation contributes to virulence difference in mice.
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