海产品中副溶血弧菌检测方法研究进展

副溶血弧菌是主要的食源性致病菌之一,主要存在于鱼虾贝等海产品中。人们在食用被副溶血弧菌感染的海产品时,容易出现腹泻、呕吐、腹痛等急性肠胃炎症状。本文主要介绍了副溶血弧菌的分子生物学、免疫学等几种快速检测方法,并对未来的发展前景做了展望。      

巢式PCR快速检测海产品中的副溶血弧菌

副溶血弧菌是一种世界范围性的食源性致病菌,食用了该菌污染的海产品可导致胃肠炎等疾病。为了建立一种可快速、特异地检测海产品中副溶血弧菌的方法,通过把副溶血弧菌基因组序列和其它不同种类弧菌的基因组序列进行比较分析,筛选出了一个副溶血弧菌特异性的标记基因-VP1331,根据该基因建立了副溶血弧菌的巢式PCR快速检测方法,并评估了其特异性、敏感性和稳定性。实验结果表明,该方法只有在以副溶血弧菌基因组DNA为模板时才能扩增出目的片段,而其它11种弧菌和非弧菌均不能扩增出目的片段。该方法的最低检测限为副溶血弧菌基因组DNA 10 fg、纯培养物6.6 CFU。人工污染实验表明,初始菌液浓度为25.7 CFU/100 mL时只需经过2 h的增菌培养即可检出。上述结果表明,VP1331基因可以作为副溶血弧菌种特异性标记,本方法可以用于污染海产品中该菌的检测与鉴定。     

海产品中副溶血弧菌的LAMP-HNB快速检测技术

采用环介导等温扩增(loop-mediated isothermal amplification,LAMP)并结合指示剂-羟基萘酚蓝(hydroxy naphthol blue,HNB)的应用,即LAMP-HNB快速检测海产品中副溶血弧菌的新方法进行了研究。首先,针对副溶血弧菌tlh基因设计特异性引物,并在反应体系中加入1μL HNB(反应浓度150μmol/L)作为反应指示剂,根据反应体系颜色变化判断反应结果,并同时将产物进行凝胶电泳验证结果,通过PCR平行实验对比,实证LAMPHNB法的反应灵敏度和特异性。结果表明,LAMP-HNB新法与PCR方法的特异性没有差异,但是,LAMP-HNB新法的灵敏度是PCR的10~100倍。经海产品实际检测验证,LAMP-HNB新法与PCR法和国标法的检测结果一致。因此,LAMP-HNB新法具有特异性强、灵敏度高、操作简便等特点,更适用于海产品处理现场的检测。     

LAMP技术快速检测海产品副溶血弧菌的条件优化

本研究以副溶血弧菌不耐热溶血素tlh为目标基因设计特异性引物,优化反应条件,并对特异性、灵敏度进行了验证。LAMP最佳反应条件为,外内引物浓度比为1∶8,Mg2+反应浓度为4 mmol/L,d NTPs浓度为3.5 mmol/L,温度为65℃,反应时长为1 h,只对副溶血弧菌产生扩增反应,与其余细菌不产生扩增反应,细菌基因组DNA和纯培养物的灵敏度约为5.45×101 fg/μL和140 cfu/m L,对人工污染食品样品进行检测,检出限为2.8×102 cfu/m L。该方法反应时间短、特异性强、灵敏度高。      

锦州笔架山海域海产品中副溶血弧菌的分子流行病学调查

为了解锦州笔架山周边海域海产品中副溶血弧菌的污染状况,实验室随机采集了蓝圆鲹等共103份常见的海产品,按照GB/T 4789.7-2008及PCR方法对副溶血弧菌进行了分子流行病学调查。结果显示,随机抽检的103份样品中,检测出含副溶血弧菌的样品38份,检出率为37%。这表明,锦州笔架山周边海域常见海产品受副溶血弧菌污染十分严重,具有极大的食品安全隐患。     

同时检测海产品中副溶血弧菌和霍乱弧菌的合检方法评价研究

目的建立水产品中副溶血弧菌和霍乱弧菌合检方法,并对合检方法对比进行评价。方法利用副溶血弧菌和霍乱弧菌阳性菌株,制备纯菌液、不同浓度梯度的人工污染样品。通过对30份纯菌液、30份人工污染样品和250份实际样品的检测,将建立的合检方法与行业标准(SN/T 1022-2010进出口食品中霍乱弧菌检验方法和SN/T 0173-2010进出口食品中副溶血性弧菌检验方法)进行比较,对合检方法进行效果评价。结果实验结果表明副溶血弧菌和霍乱弧菌合检方法,与行业标准检测结果完全一致。结论该方法可靠,对实验仪器和操作人员的要求低,具有良好的实用性,适合基础检测实验室。     

北戴河海产品中副溶血弧菌的毒力、耐药性和遗传特征

为了解北戴河地区水产品中副溶血弧菌的毒力基因携带现况、耐药性和遗传特征,在2021年8月分析来自北戴河地区3家食堂和自采的26份海鲜样本,检出率为38.46%。对从中分离鉴定的10株副溶血弧菌通过PCR筛选4种主要毒力基因（tdh、trh、tlh、toxR）,利用BD自动微生物鉴定和药敏分析系统,并对分离株做二代测序,基于核心SNP位点构建系统发育树,耐药基因与毒力基因携带情况。结果表明,（菌株tlh、toxR基因携带率均为100%）,所有分离副溶血弧菌均不携带tdh基因或trh基因;抗生素氨曲南耐药率为100.00%、对氨苄西林中介耐药率为100.00%,对其余16种抗生素均敏感;10株分离株都含β-内酰胺类耐药基因CARB-18,这与其耐药表型相对应;一株分离株含有毒力基因mexE,其在同类研究中少有报道;共鉴别出9种不同的序列类型;食堂分离的部分菌株与当地海洋生物分离株同源性较高,部分菌株与纳入分析的国外菌株同源性较高。上述分析提示,当地副溶血弧菌对人类健康构成潜在威胁,其来源复杂,具有高度遗传多样性,可能存在国外副溶血弧菌污染源,需加强监测。     

基于弧菌毒力基因多重PCR法的建立及其在副溶血弧菌毒力基因检测中的应用

针对副溶血弧菌常见的11种毒力基因(tox R、Collagenase、tox S、trh、tdh、tlh、Ure R、Fla A、omp W、Asp A、fur),建立了两套六重PCR检测体系,应用于副溶血弧菌环境分离株和水产品分离株的毒力基因分布情况调查。在调查的248株副溶血弧菌中,鞭毛丝蛋白基因Fla A、外膜蛋白基因omp W和铁吸收调节蛋白基因fur的分布最广(100%),其次为碱性丝氨酸蛋白酶基因Asp A(99.60%),胶原蛋白酶基因Collagenase、不耐热性溶血毒素基因tlh以及毒力调控基因tox R和tox S的分布率均在90%以上且tox R和tox S的分布极为相似,尿素酶基因Ure R的分布极少(1.21%),而耐热直接溶血素基因tdh和耐热相关溶血素基因trh在这248株副溶血弧菌中没有检出。本研究建立的多重PCR检测体系能快速、高效地检测多个毒力基因的分布情况,为副溶血弧菌的毒力机制研究和风险评估提供方法和依据。     

海产品中副溶血性弧菌检测方法研究进展

副溶血性弧菌是一种重要的食源性致病菌,已经成为我国食源性疾病暴发事件的重要致病因素之一。目前该菌的检测方法较多,但各有利弊。本文就现有的检测手段进行总结,以便为其检测方法的应用和发展提供参考。     

水产品中副溶血性弧菌LAMP检测方法的优化

本文用副溶血性弧菌不耐热溶血素（tlh）基因作靶标,优化环介导等温扩增技术检测水产品副溶血性弧菌的方法。体系用Bst DNA聚合酶催化,恒温反应60 min,产物分别用2%琼脂糖凝胶电泳和SYBR Green I染色鉴定,对各项反应参数进行优化;将新鲜菌液作10倍梯度稀释后进行LAMP和PCR反应,比较二者的敏感度;对32株食源性病原菌（包括分离于水产品的25株副溶血弧菌,1株副溶血性弧菌ATCC17802标准株,大肠杆菌D5、金黄色葡萄球菌CMCC26003、志贺氏菌B4、蜡样芽胞杆菌63302、单核细胞增生李斯特菌54001和沙门氏菌50041各1株）进行LAMP扩增,验证其特异性;虾样品用菌液进行模拟污染,分析LAMP的可靠性。各参数最佳条件为：Mg2+浓度为3.6 mmol/L,dNTPs浓度为0.96 mmol/L,Bst DNA聚合酶用量为4.8 U,内外引物浓度比为8:1,最佳反应温度为63 ℃,时间为60 min;LAMP的检测限为1 CFU/mL,低于PCR方法的最低检测限;特异性试验检测26株副溶血性弧菌均为阳性,6株非副溶血性弧菌为阴性;人工污染试验中检测限达1 CFU/mL,无假阳性。建立的LAMP方法操作简单且灵敏度高,适用于水产食品中副溶血弧菌的现场快速检测。     

副溶血性弧菌和溶藻弧菌的双重PCR检测技术

主要针对副溶血性弧菌(Vp BJ1997)和溶藻弧菌(Va ATCC17749)的gyrB基因的不同位点设计特异性引物,利用双重PCR技术同时检测Vp(BJ1997)和Va(ATCC17749),并对该反应体系的特异性以及灵敏度进行检测。结果显示Vp(BJ1997)灵敏度的检测下限可达2×10~2CFU/mL,Va(ATCC17749)灵敏度的检测下限可达2.03×10~2CFU/mL,双重PCR与单一PCR检测的灵敏度的数量级是相同的;与沙门氏菌(ATCC27892)、金黄色葡萄球菌(ATCC13565)、大肠杆菌(35218)、河弧菌(H265)、鳗弧菌(M936)、创伤弧菌(ATCC27562)无交叉反应;在人工污染试验中,混合菌液的检测下限2.84×10~3CFU/mL,而进一步稀释至2.84×10~2CFU/mL时,Vp(BJ1997)已检测不出,Va(ATCC17749)仍可检出。研究表明,该方法特异性强,灵敏度高,操作简单,成本低,检测速度快,为基层单位同时检测副溶血性弧菌和溶藻弧菌提供了一种快速准确的方法,对控制水产品中这两种致病性弧菌具有重大的意义。     

Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA

PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V. parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.     

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.     

食品中副溶血性弧菌PCR快速检测方法的研究

为建立食品中副溶血性弧菌 (VP)的PCR检测方法 ,选取tl基因作为靶序列设计一对引物 ,用该引物对 14株从国内食品中分离的副溶血性弧菌 (经传统方法验证 )和 30株非副溶血性弧菌进行PCR扩增 ,并用此方法对人工污染食品进行检测。扩增片段表现出极好的特异性 ,对人工污染的冷冻虾仁、沙丁鱼的检出限为 10CFU g ,且与传统方法结果吻合。该方法适宜于食品中副溶血性弧菌的检测。     

Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus.     

副溶血弧菌快速检测技术研究进展

副溶血弧菌是一种重要的嗜盐性食源性致病菌,广泛存在于近海岸的海水、海底沉积物以及鱼虾、贝类等海洋生物中。在我国沿海地区,由副溶血弧菌引起的食物中毒已成为微生物中毒的首要因素。本文分别以副溶血弧菌菌体、核酸、蛋白质和代谢产物为检测对象,介绍了各种PCR技术、酶联免疫吸附技术和生物传感器技术等目前副溶血弧菌的热点快速检测技术,对各种技术进行了比较分析,并对未来的发展前景作了展望。      

Pathogenetic characterization of Vibrio parahaemolyticus isolates from clinical and seafood sources

A total of 216 Vibrio parahaemolyticus isolates from seafood and clinical samples in eastern China were investigated for their hemolytic and urea-producing phenotypes, presence of putative virulence genes tdh and trh. Twenty-one clinical isolates (84%, 21/25) and 3 seafood isolates (1.57%, 3/191) were tdh-positive while only 3 clinical isolates (12%) and 7 seafood isolates (3.66%) were positive for trh gene. We further examined the pathogenicity of selected V. parahaemolyticus isolates in in vitro and in vivo systems. The clinical isolates were apparently more enteropathogenic (74.26 per thousand vs 62.07 per thousand expressed as intestine/body weight ratio, P<0.01) and more virulent than their seafood counterparts to mice (log LD(50) 6.86 vs 7.40 via orogastric route, P<0.05). They were also more adherent to in vitro cultured cells and of higher cytotoxicity as measured by LDH release of the HeLa cells although there were no statistical differences. The tdh-positive V. parahaemolyticus isolates were of higher enteropathogenicity (P<0.05, 74.24 per thousand vs 60.55 per thousand) and more virulent (log LD(50) 6.55 vs 7.21 via intraperitoneal route, P<0.05) than tdh-negative isolates. The tdh-positive isolates were generally more cytotoxic and adhesive to the cultured cell lines as well. From the in vitro and in vivo pathogenicity profiles, trh-positive isolates seemed to line between tdh-positive isolates and those without tdh and trh. There were two isolates H8 and H10 from clinical cases having moderate enteropathogenicity and virulence to mice, but were tdh-negative yet trh-positive. These results seem to suggest that hemolysins TDH and/or TRH may not be necessarily the only virulence factors of pathogenic V. parahaemolyticus isolates.     

水产品中副溶血性弧菌特异性二重PCR检测方法的研究

建立快速检测水产品中副溶血性弧菌(Vibrio parahae-molyticus)的二重PCR方法.以副溶血性弧菌特异性基因tlh和toxR为靶基因,选择2对引物,对5株副溶血性孤菌和40株非副溶血性弧菌进行特异性检测;梯度稀释副溶血性弧菌基因组DNA,以不同稀释度DNA作PCR扩增;在鱼肉样品中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增;应用该方法对实际样品进行检测.以tlh和toxR为靶基因的两对引物对副溶血性弧菌的检出有很好的特异性.PCR检测的灵敏度在DNA水平上达到28.76 pg;人工污染样品,当起始污染量为1 CFU/mL时,37℃增菌培养10 h即可检出.本试验一共检测了21份水产品样品,有14份检出了副溶血性弧菌.     

牦蛎食品中副溶性弧菌的分离与鉴定

从出口水产食品牦蛎样品中检测副溶血性弧菌.应用细菌形态学观察、嗜盐性试验、细胞色素氧化酶试验、靛基质试验、赖氨酸脱羧酶试验、精氨酸双水解酶试验、V-P试验、蔗糖分解试验、O/F试验等鉴定疑似副溶血性弧菌菌株.结果表明经相关试验证明,从牦蛎样品分离出的菌株为副溶血性弧菌.结论从水产食品牦蛎中分离出2株副溶血性弧菌.     

Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of seafood samples naturally contaminated with Vibrio parahaemolyticus. A total of 171 seafood samples enriched in alkaline peptone water (APW) were assessed by LAMP assay and conventional culture methods, which consist of a combination of APW enrichment culture and plating onto CHROMagar Vibrio and TCBS agars. Compared with V. parahaemolyticus isolation using the conventional culture test, LAMP results showed 100% (30/30) and 90.8% (128/141) sensitivity and specificity, respectively. The conventional culture test required more than 3 days to isolate and identify V. parahaemolyticus in the APW enrichment culture. In contrast, the LAMP assay was markedly faster, requiring less than 60 min from the beginning of DNA extraction to final detection of V. parahaemolyticus. In total, the LAMP assay required 17-19 h from the beginning of enrichment culture to final determination. This is the first report of the LAMP assay for rapid screening of seafood samples naturally contaminated by V. parahaemolyticus.