基于toxR基因病原哈氏弧菌PCR检测方法的建立

基于toxR基因序列设计检测哈氏弧菌的1对特异性引物,通过PCR反应条件优化、PCR反应特异性及敏感性检测,建立一种哈氏弧菌的特异性分子检测方法.试验结果表明,该引物可使哈氏弧菌扩增出大小为390 bp的toxR基因片段,3种水产动物病原弧菌未扩增出任何条带,敏感性检测结果为该PCR反应最低能检测出0.375 ng/μ...     

Identification of Vibrio parahaemolyticus in Seafood by Multiplex PCR

ABSTRACT

Vibrio parahaemolyticus is a human pathogen frequently found in seafood. Once the seafood is contaminated by V. parahaemolyticus, it can become a vehicle for foodborne illness. The conventional culture methods for detection of V. parahaemolyticus are time-consuming and cannot differentiate pathogenic strains from nonpathogenic ones. In this study, a multiplex polymerase chain reaction (PCR) technique was investigated for detecting tdh, chiA, and toxR of V. parahaemolyticus. The sensitivity of the multiplex PCR was determined by testing 28 strains of V. parahaemolyticus, 15 non-V. parahaemolyticus strains, and fresh seafood spiked with cells of V. parahaemolyticus. All the strains were analyzed for production of thermostable direct hemolysin (TDH) and chitinase. This study showed that both the chiA and toxR are excellent markers for detecting V. parahaemolyticus strains, and a multiplex PCR targeting chiA and tdh genes can be applied to simultaneously detect environmental and pathogenic V. parahaemolyticus.     

宁波地区贝类产品中副溶血弧菌的分离与鉴定

从2007年3月至6月,在宁波市5个不同的农贸市场共采集贝类样品360份,分离菌株71株,其中5株代表菌株革兰氏染色阴性、弧状、具极生单鞭毛,利用葡萄糖、甘露醇产酸,不利用乳糖、蔗糖,精氨酸双水解酶阴性,VP试验为阴性,赖氨酸脱羧酶、鸟氨酸脱羧酶为阳性,靛基质、甲基红试验阳性,不产生H2S,初步判断为副溶血弧菌;进一步进行了基于16S rRNA和toxR序列的PCR检测,序列测定结果验证了该5株细菌确实为副溶血弧菌.以16S rRNA序列为基础,分析了分离株与相关细菌菌株的同源性,构建了系统发育树.     

牡蛎中副溶血弧菌荧光定量PCR检测方法的建立及其应用

以副溶血弧菌毒素调控基因(toxin regulations,toxR)作为靶标基因,设计特异性引物及TaqMan探针,以含toxR基因的质粒为模板,建立质粒拷贝数与CT值的标准曲线,分别采用含toxR基因质粒、纯培养的副溶血弧菌和添加副溶血弧菌的牡蛎(Ostrea)模拟样品进行灵敏度试验,结果表明,其灵敏度分别为15拷贝、18 CFU/mL和180CFU/mL.同一个样品的30次重复性试验表明,试验内及试验间的变异系数分别为0.95%和1.5%.结果显示,本研究建立的副溶血弧菌荧光定量PcR检测方法特异性强、灵敏度高、重复性好,可用于牡蛎等水产品中副溶血弧菌的定量检测.     

副溶血弧菌主要毒力因子三重荧光定量PCR检测方法的建立与应用

为建立快捷、准确、高效检测副溶血弧菌的方法,以副溶血弧菌种属特异性基因toxR和毒力因子tdh、trh基因序列,分别设计3对特异性引物及相应TaqMan探针,并在toxR、tdh和trh基因的3个探针的5'端分别标记FAM、HEX、CY5荧光报告基团,3'端标记BHQ1淬灭荧光基团,通过优化反应体系和反应参数,确定最佳反应体系,建立一种基于Taq Man探针定量检测副溶血弧菌的三重荧光定量PCR方法。结果:本试验所建立三重荧光定量PCR方法与其他菌株无交叉反应,其最低检出限达到了10 CFU/m L,重复性试验中每组变异系数均小于1%;检测人工染菌的虾肉和贝类样品时,最低检出限亦达到10 CFU/m L,且整个检测扩增时间大约为1 h。结果表明,本研究建立的三重荧光PCR检测方法,特异性强、敏感性高、重复性好且耗时短,是高通量检测致病性副溶血弧菌的有效手段。     

缢蛏副溶血弧菌的分离与鉴定

从患病缢蛏中分离纯化到一株弧菌,用形态学、生理生化指标以及16SrRNA、toxR基因的PCR扩增等方法,鉴定为副溶血弧菌(Vibrio Parahaemolyticus,VP)。     

A novel multiplex PCR method for detecting virulent strains of Vibrio alginolyticus

The bacterial strains obtained from various origins were tested with the novel primers targeting the collagenase gene, ompK gene and toxR gene to establish a multiplex polymerase chain reaction (PCR) method. These primers successfully recognized all virulent strains of Vibrio alginolyticus, but the avirulent strains were not recognized by the multiplex PCR because of lack of the collagenase and toxR genes. In a 50 μL multiplex PCR mixture, the lowest detection limit is 8.8 × 10² cells of virulent strains of V. alginolyticus. The multiplex PCR method was successfully developed to identify virulent strains of V. alginolyticus, and provides a rapid, sensitive, specific and reliable technology for diagnosing virulent strains of V. alginolyticus. Therefore, the novel multiplex PCR in the present paper can be useful for any laboratory working with vibriosis detection of aquatic animals.     

致病性副溶血性弧菌特异性三重PCR检测方法研究

[目的]建立同时检测致病性副溶血性弧菌gyrase、tdh、toxR基因的三重PCR快速检测方法。[方法]用3种基因的特异性引物分别对副溶血性弧菌ATCC33847的模板DNA进行单一扩增,找到各自引物最佳扩增条件;再用3种引物同步对模板DNA进行扩增,通过优化引物浓度、引物间比例以及退火温度,建立最佳扩增体系。[结果]在最佳三重PCR反应条件下,gyrase、tdh和toxR能同时扩增出清晰条带,大小分别为91、269和368 bp。[结论]该研究为致病性副溶血性弧菌的快速检测提供了一种新的技术方法。     

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.     

副溶血性弧菌的检测研究

[目的]对toxR进行副溶血性弧菌特异性检测,探讨toxR靶基因能否准确检测副溶血性弧菌,从而消除其他研究者对该基因特异性的疑虑。[方法]采用SN/T1870—2016中副溶血性弧菌toxR的引物探针对副溶血性弧菌和其亲缘关系接近的弧菌标准菌株进行检测。[结果]采用实时荧光PCR方法证实该引物探针只能扩增出副溶血性弧菌,其他弧菌诸如溶藻弧菌、创伤弧菌、霍乱弧菌等未得到扩增。[结论]证实SN/T1870—2016中toxR的引物探针特异性高。该研究可为各检测机构提供数据支持,为副溶血性弧菌的检测与研究奠定更为坚实的基础。