Levels of detection of Cryptosporidium oocysts in mussels (Mytilus galloprovincialis) by IFA and PCR methods

Cryptosporidium spp. are monoxenous protozoan parasites that cause gastrointestinal diseases in humans and animals. Shellfish harvesting areas can become contaminated by the infectious stage of the parasite and humans are therefore at risk of infection either by consumption of shellfish, or by taking part in recreational activities in these areas. In the present study we determined the levels of detection, by IFA and PCR techniques, of Cryptosporidium oocysts in mussels experimentally contaminated with a theoretical number of oocysts. There was a significant correlation between the results obtained by both techniques (P<0.05). IFA and PCR were also applied to a total of 222 samples of mussels (Mytilus galloprovincialis) destined for human consumption. In the naturally contaminated samples, we detected a 31.1% of contamination and only Cryptosporidium parvum (previously denominated C. parvum genotype II) was identified.     

生物素-亲和素斑点酶联免疫吸附试验快速检测鱼源副溶血性弧菌的研究

本研究建立了快速检测鱼源副溶血性弧菌的BA-Dot-ELISA方法.用该法可检出每毫升接种10个菌,增菌18h的培养物.该法敏感性为10~4个/mL,比Dot-ELISA敏感10～100倍.不与溶藻性弧菌、亲水气单胞菌等13种杂菌发生交叉反应.增菌前杂菌多于副溶血性弧菌10~6时,对检测结果有影响.可在22～24h报告结果,比常规培养法快4～6d以该法对带鱼、小黄花鱼、马面鱼、鲤鱼等311份样本进行检测,检出率分别为47.0%、41.7%、14.8%和11.8%,与常规培养法相差不显著(P>0.05).生化试验复检结果表明,检出的阳性可凝菌株均为副溶血性弧菌.该法适用于在短时间内对大批样本的检测.     

副溶血性弧菌PCR检测方法的建立

为了建立适于基层单位应用的针对副溶血性弧菌检测的特异PCR方法,检测副溶血性弧菌的基因序列.针对该菌的属特异性基因t1基因进行引物设计,扩增片段大小为449 bp.运用该PCR法可特异性的从副溶血性弧菌ATCC17802标准株中扩增出目的基因片段,与GenBank上发表的序列同源性为100%.而经常污染海产品的溶藻弧菌,嗜水气单胞茵标准株J-1,铜绿假单胞茵标准株ATCC27853结果均为阴性.该PCR法最低检出菌体DNA量为10-2ng以及最低检出菌数为5.7 × 103 CFU/mL.对临床上送检的35份样品进行菌体DNA PCR方法检测并同时与国标GB/T4789.7-2003中使用的检测方法进行比对,两种检测方法的结果完全符合,与国标中使用的检测方法相比可大大缩短鉴定时间.因此该PCR法能快速鉴定当前副溶血性弧菌流行群,有利于流行病学溯源等研究.     

用PCR检测副溶血性弧菌耐热溶血素基因

根据副溶血性弧菌的耐热溶血素基因（ｔｄｈ）设计了２对引物，经聚合酶链式反应（ＰＣＲ）检测，所有１８株神奈川试验阳性副溶血性弧菌均呈阳性反应，而６株神奈川试验阴性副溶血性弧菌呈阴性反应，其他９种（３４株）弧菌和８株其他革兰氏阴性菌也呈阴性反应。检测敏感性可达２ＣＦＵ的副溶血性弧菌。     

Development of Real-time PCR Method for Rapid Detection of Vibrio parahaemolyticus

To establish a rapid assay for detection of Vibrio parahaemolyticus(VP), Real-time PCR method was developed targeting to toxR gene of Vibrio parahaemolyticus.The results showed that the test for 15 bacteria strains using the Real-time PCR method, only Vibrio parahaemolyticus test was positive, indicating that the method had high specificity.In addition, the sensitivity of Real-time PCR was 4.9 CFU/mL.Furthermore, a total of 3 positive samples for Vibrio parahaemolyticus were detected from 150 clinical samples by the Real-time method, which was in accordance with the testing result by GB 4789.7-2013 standard detection protocol.Therefore, the Real-time method provided a novel rapid and sensitive detection method with good practicality for Vibrio parahaemolyticus infection.     

副溶血弧菌实时荧光定量PCR快速检测方法的建立

为建立检测副溶血弧菌(Vibrio parahaemolyticus,VP)的快速检测方法,本研究以VP toxR基因为靶基因设计合成引物及TaqMan探针,建立了实时荧光定量PCR快速检测VP的方法。结果显示,对15株试验菌株进行实时荧光定量PCR检测,只有VP检测为阳性,表明该检测方法特异性强;该方法的灵敏度为4.9 CFU/mL,利用该检测方法对采集的150份样品进行检测,共计检出3份VP阳性样品,与国标法(GB 4789.7-2013)检测结果一致,显示了良好的实用性。该检测方法灵敏度高、特异性强,具有良好的实用性。     

副溶血性弧菌分离鉴定

利用TCBS琼脂培养基和科玛嘉弧菌显色培养基对细菌进行分离,采用糖分解试验、氧化酶试验、IMVi试验等生化试验鉴定海产品中副溶血性弧菌,为防治疾病提供依据。     

A Marteilia-like parasite in blue mussels Mytilus edulis in China

Species of the genus Marteilia (Phylum Paramyxea) are protozoan parasites of marine mollusks. Marteilia spp. have been detected in mollusks from different parts of the world, but the presence of these parasites in China has not been previously reported. Therefore, a survey was conducted to look for the presence of Marteilia spp. in blue mussels Mytilus edulis and Asian green mussels Perna viridis collected along China's coasts. Histological and PCR analyses revealed that 5 of 180 M. edulis (prevalence = 2.8%) were positive for infection with a Marteilia-like organism, whereas the parasite was not detected in any of the 80 P. viridis individuals tested. Total genomic DNA was extracted from the infected tissue sections for PCR amplification. The PCR amplification with Marteilia primers SS1 and SAS1 yielded the expected 641-bp product. Sequencing results showed that the 18S ribosomal RNA gene fragment from the protozoans found in M. edulis from China was 88% similar to that of Marteilia refringens, a species that was reported from M. edulis and European flat oysters Ostrea edulis collected in France. This is the first report of a Marteilia-like organism infecting M. edulis in China.     

副溶血性弧菌耐药基因的研究进展

副溶血性弧菌为水产品主要致病菌之一,水产业和农业中抗生素的过量使用导致副溶血性弧菌对所推荐使用的抗生素产生了抗性,并且在副溶血性弧菌中已经发现了质粒和整合子等基因移动元件,更增加了与其他细菌发生耐药基因相互交换和重组的几率。对副溶血性弧菌的耐药研究现状、耐药机制和耐药基因的水平转移进行综述。     

海产品中副溶血弧菌的分离与鉴定

副溶血弧菌（Vibrio Paraaemolyticus)是一种致病性嗜盐菌，常引起人类食物中毒，通过对四种不同海产品的检测，发现该菌检出率很高，其中，海虾中的检出率高达90％，海产鱼为70％，贝类为60％，海蟹为40％，平均检出率为65％，动物试验证实，该菌致病性较强。     

The detection of Vibrio parahaemolyticus and Vibrio alginolyticus using modified culture media

Studies have been completed for the detection of Vibrio(V.) parahaemolyticus and V. alginolyticus in Baltic Sea fish. The author had prepared for that purpose a liquid culturing medium and a solid substrate. The percentage of positive findings is remarkable. The results so far recorded should be a good reason for further studies.     

副溶血弧菌和霍乱弧菌双重荧光定量PCR快速检测方法的建立与应用

建立一种基于TaqMan探针法同步定量检测副溶血弧菌和霍乱弧菌的双重荧光定量PCR方法。根据副溶血弧菌toxR基因和霍乱弧菌ompW基因设计特异性引物与TaqMan探针。toxR和ompW探针5'端分别标记FAM、CY5荧光报告基团,3'端均标记BHQ1荧光淬灭基团。结果显示:该方法的副溶血弧菌和霍乱弧菌最低检测限均达到10 CFU/m L,灵敏度高;特异性试验表明这两种细菌与其他病原菌(大肠杆菌O157、沙门氏菌、拟态弧菌、创伤弧菌)无交叉反应;批间和批内重复性实验表明变异系数均小于1.5%,说明重复性好。采用人工染菌虾肉样品,副溶血弧菌和霍乱弧菌的最低检测限分别为100 CFU/m L和50 CFU/m L,人工染菌贝类样品的最低检测限分别为50 CFU/m L和50 CFU/m L。两种细菌在虾肉中富集2 h后的最低检出限均为1 CFU/m L。结论:本研究所建立的双重荧光PCR检测方法具有灵敏度高、特异性强和重复性好的特点,包括DNA提取的整个检测过程可在1至3 h内完成,是同时快速检测副溶血弧菌和霍乱弧菌的有效手段。     

Flash flood simulation and valve behavior of Mytilus galloprovincialis measured with Hall sensors

Mussels close their shell as a protective strategy and the quantification of this behavioral marker may represent an alarm signal when they are exposed to environmental stressors. In the present study, we investigated the ability of the Mediterranean mussel Mytilus galloprovincialis to recover and then the resilience or inertia of valve activity after a pulsing exposition to diverse levels of salinity (5, 10, 20, and 35 PSU as reference value). The trial simulated an event of drastic and sudden reduction of seawater salinity thus mimicking an event of flash flood from intense rain. Valve gaping and movements were measured in continuous cycle for 10 days using a customized magneto-electric device which uses Hall sensors. Results showed that under normal conditions of salinity (35 PSU), the general pattern of valve movements was a continuously open state with sporadic spikes indicating a closing motion. At salinity of 5, PSU mussels reacted by closing their valves, leading to a 77% mortality on the 4th day. At salinity of 10, PSU animals were observed with closed valves for the entire duration of the exposure and no mortality occurred, they showed a significant reduction in the valve activity once the reference value of salinity was re-established. In contrast, salinity of 20 PSU did not trigger a significant behavioral response. Interestingly, there no define rhythms of valve movements were recorded during salinity challenges.     

Attachment of Vibrio parahaemolyticus strains to estuarine algae.

Attachment of Vibrio parahaemolyticus strains to estuarine microalgae was examined in artificial seawater by viable counts of the organism and direct counts of the bacterial cells after immunoperoxidase staining. Thermostable direct hemolysin (TDH)-producing and TDH-non-producing strains of V. parahaemolyticus were found to attach to five estuarine strains of Navicula (diatom alga) in similar levels. The level of the bacterial attachment depended on salinity and temperature of the water, in which the maximum attachment was observed in 15% artificial seawater at 25 degrees C, a typical condition of Hashizu estuary in Japan during summer months. The attachment was inhibited by pectinase digestion of the algal cells. These evidences confirmed the participation of the microalgae to the ecological cycle of V. parahaemolyticus at the estuary.     

水生动物中分离的副溶血性弧菌菌株分子分型研究

本研究通过采用核糖体分型RT、脉冲场凝胶电泳PFGE、多位点序列分型MLST三种方法,对从水生动物中分离到的59株副溶血性弧菌进行分子分型研究。结果将59株副溶血性弧菌分为43个RT型,9个大的聚类群,DI为0.9754;51个PFGE型,13个大的聚类群,DI为0.9988;53个ST型,包括107个新的等位基因、46个新的ST型,DI为0.9982。结果显示PFGE的分辨力最高,MLST较PFGE稍低,RT的分辨力最低。总体来看59株副溶血性弧菌菌株间亲缘关系较远,从同一年份、同一地区、同类样品中分离到的菌株被分为不同的型,表现出较大的遗传多样性。     

副溶血弧菌海产品分离株及临床分离株的多位点序列分型

选取毒力调控基因toxRS和看家基因gyrB、recA作为靶基因,对浙江沿海地区40株副溶血弧菌海产品分离株与8株临床分离株进行多位点序列分型。toxRS的多态性位点比例(10.2%)虽低于gyrB(12.0%)与recA(25.4%),但与gyrB均可分辨出最多的序列型(38),具有最强的分辨力(0.986)。3个基因串联后可分出44个序列型,分辨力达0.994。副溶血弧菌分离株呈现出较大的多样性。各地的海产品分离株分布于A群、B群,而临床分离株则主要集中于A群;C群仅包括1个临床分离株。其中临床分离株C2、C5、C7与海产品分离株F24属于同一个序列型,由此可推测该序列型在地域上分布较为广泛并可引起人发生胃肠炎等。因而副溶血弧菌所引起的对公共卫生的潜在风险性不容忽视。     

依赖于核酸序列恒温扩增技术快速检测副溶血性弧菌方法的建立

为建立副溶血性弧菌的快速检测方法,本研究采用依赖于核酸序列恒温扩增(NASBA)技术,以副溶血性弧菌的tlh基因为扩增的靶基因设计特异性引物和探针,建立可快速扩增副溶血性弧菌的NASBA方法.特异性和灵敏度试验结果表明:该NASBA方法对副溶血性弧菌的最小检出量为5.1×102 cfu/mL,高于普通PCR方法,而且与其他种属的菌无任何交叉反应.此外,本研究将副溶血弧菌扩增产物采用通用型核酸扩增物快速检测板进行检测,实现了特异性强的快速副溶血弧菌的检测.该方法对仪器要求低,具有良好的应用前景.