食源性变形杆菌属PCR检测方法的研究
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摘要
针对食源性变形杆菌属食物中毒事件逐年上升的趋势,研究建立新型快速灵敏的变形杆菌属检测鉴定方法。通过建立3种常规PCR方法和1种荧光PCR方法,对66株变形杆菌属鉴定,与传统分离培养法和全自动微生物分析系统检测法进行比较研究,结果如下:
根据1961-2007年发表的218篇关于264起变形杆菌属食物中毒案例分析,变形杆菌属食物中毒自二十世纪的80年代到90年代到目前的21世纪,呈现上升趋势,广东省和山东省为变形杆菌属食物中毒多发省份,绝大多数中毒事件都发生在夏秋季节,以城市中的饮食服务单位居多,中毒食品多为动物性食品,尤其是熟食制品,中毒菌种主要是奇异变形杆菌和普通变形杆菌。在187起变形杆菌属食物中毒的检测方法中,94.65%采用传统分离培养法,其余5.35%采用全自动微生物分析系统法,没有任何采用PCR及其它分子生物学检测方法的报道。
传统分离培养法和全自动微生物分析系统检测法对66株样本分离菌株的鉴定结果全部为阳性,鉴定结果与中山大学附属第三医院和暨南大学附属第一医院菌种鉴定保存的结果一致,符合率100%,验证了变形杆菌属菌种的真实性。
选取atpD基因和tuf基因作为靶序列,设计了3对特异性引物,分别建立了3种相应的检测变形杆菌属的常规PCR法,对3株变形杆菌属标准菌株、66株样品分离株及13株非变形杆菌属菌株进行扩增实验,结果显示,3种常规PCR法对3株标准菌株和66株样品分离株的检测结果均为阳性,13株非变形杆菌属菌株检测结果均为阴性。鉴定结果与传统分离培养法和全自动微生物分析系统检测法结果完全一致,但检测时间仅为6-8 h,其检测速度、灵敏度和特异性表现出独特的优势。
基于atpD基因设计1对特异性引物,建立了1种SYBR GreenⅠ荧光PCR法,用于检测变形杆菌属,其鉴定结果与常规PCR方法完全一致,但本方法所耗时间仅为1-2 h,表现出更优的特异性、重现性和灵敏度,并且操作更为简便。
对8条变形杆菌属菌株atpD基因测序,其结果与Genbank中变形杆菌属atpD基因序列不同,本文所测基因序列是截至目前为止尚未公开的新的变形杆菌属atpD基因序列。
综上所述,本文所建立的3种常规PCR方法和1种荧光PCR方法都可作为快速检测鉴定变形杆菌属的可选方法。
Based on the increasing trend of food-borne Proteus poison, detection methods with rapidity and sensitivity should be set up for control of this bacterium. Three types of conventional polymerase chain reaction (PCR) methods and one type of newly fluorescent real-time PCR were developed to detect and identify the sampled 66 species of Proteus by comparing with the traditional plate-culture and the Vitek auto microbe system methods. Results obtained are shown as followings:
Food-borne illnesses study from 264 cases reported by 218 papers published during 1962-2007 in China showed that the number of food infection via Proteus went up gradually from 1980s, 1990s to the present 21st century, with the two dominant provinces Guangdong and Shandong, two peaks of prevalence seasons the summer and the autumn, target of animal food, particularly cooked meat products, caused mainly by Proteus mirabilis and Proteus vulgaris. Detection methods used for Proteus in the reported 187 cases were traditional plate-culture method and Vitek auto microbe system which accounted for 94.65% and 5.35%, respectively. No any report of using PCR as a detection method to identify Proteus.
Samples of 66 isolated Proteus were detected by plate-culture and Vitek auto microbe system, respectively, and the results were both consistent with the report from the samples donors the third Affiliated Hospital of Sun Yat-Sen University and the first Affiliated Hospital of Jinan University, indicating that the authenticity of those Proteus strains were proved.
There pairs of primers for conventional PCRs were designed by taking the aptD and tuf genes as objective sequences to amplify the corresponding genes of 3 references Proteus, 66 isolated sample Proteus and 13 non-Proteus strains under the various PCR conditions. Results showed that 3 references Proteus and 66 isolated sample Proteus strains were all positive whereas 13 non-Proteus strains negative, which were exact consistent with the results detected by the conventional methods. However, the newly PCR methods took only 6-8 h, and appeared advantages of rapidity, concise, sensitivity and specificity.
One specific primer specified for SYBR Green I fluorescent real-time PCR, based on atpD gene, were designed for rapid detection of Proteus. The tested results were exact same as the previous conventional PCR methods. However, the time spent for this detection was only 1-2 h, much more quick, specific, sensitive, simple than the conventional methods.
Sequences of 8 bands of aptD genes tested results showed that there are no such sequences of Proteus aptD genes in the existed GenBank. Therefore, the sequences of Proteus aptD genes tested in this study are the first report in the world.
In conclusion, the three conventional PCR methods and one specific fluorescent real-time PCR method are alternative reliable and rapid method applied for the rapid detection and identification of Proteus in food.
根据1961-2007年发表的218篇关于264起变形杆菌属食物中毒案例分析,变形杆菌属食物中毒自二十世纪的80年代到90年代到目前的21世纪,呈现上升趋势,广东省和山东省为变形杆菌属食物中毒多发省份,绝大多数中毒事件都发生在夏秋季节,以城市中的饮食服务单位居多,中毒食品多为动物性食品,尤其是熟食制品,中毒菌种主要是奇异变形杆菌和普通变形杆菌。在187起变形杆菌属食物中毒的检测方法中,94.65%采用传统分离培养法,其余5.35%采用全自动微生物分析系统法,没有任何采用PCR及其它分子生物学检测方法的报道。
传统分离培养法和全自动微生物分析系统检测法对66株样本分离菌株的鉴定结果全部为阳性,鉴定结果与中山大学附属第三医院和暨南大学附属第一医院菌种鉴定保存的结果一致,符合率100%,验证了变形杆菌属菌种的真实性。
选取atpD基因和tuf基因作为靶序列,设计了3对特异性引物,分别建立了3种相应的检测变形杆菌属的常规PCR法,对3株变形杆菌属标准菌株、66株样品分离株及13株非变形杆菌属菌株进行扩增实验,结果显示,3种常规PCR法对3株标准菌株和66株样品分离株的检测结果均为阳性,13株非变形杆菌属菌株检测结果均为阴性。鉴定结果与传统分离培养法和全自动微生物分析系统检测法结果完全一致,但检测时间仅为6-8 h,其检测速度、灵敏度和特异性表现出独特的优势。
基于atpD基因设计1对特异性引物,建立了1种SYBR GreenⅠ荧光PCR法,用于检测变形杆菌属,其鉴定结果与常规PCR方法完全一致,但本方法所耗时间仅为1-2 h,表现出更优的特异性、重现性和灵敏度,并且操作更为简便。
对8条变形杆菌属菌株atpD基因测序,其结果与Genbank中变形杆菌属atpD基因序列不同,本文所测基因序列是截至目前为止尚未公开的新的变形杆菌属atpD基因序列。
综上所述,本文所建立的3种常规PCR方法和1种荧光PCR方法都可作为快速检测鉴定变形杆菌属的可选方法。
Based on the increasing trend of food-borne Proteus poison, detection methods with rapidity and sensitivity should be set up for control of this bacterium. Three types of conventional polymerase chain reaction (PCR) methods and one type of newly fluorescent real-time PCR were developed to detect and identify the sampled 66 species of Proteus by comparing with the traditional plate-culture and the Vitek auto microbe system methods. Results obtained are shown as followings:
Food-borne illnesses study from 264 cases reported by 218 papers published during 1962-2007 in China showed that the number of food infection via Proteus went up gradually from 1980s, 1990s to the present 21st century, with the two dominant provinces Guangdong and Shandong, two peaks of prevalence seasons the summer and the autumn, target of animal food, particularly cooked meat products, caused mainly by Proteus mirabilis and Proteus vulgaris. Detection methods used for Proteus in the reported 187 cases were traditional plate-culture method and Vitek auto microbe system which accounted for 94.65% and 5.35%, respectively. No any report of using PCR as a detection method to identify Proteus.
Samples of 66 isolated Proteus were detected by plate-culture and Vitek auto microbe system, respectively, and the results were both consistent with the report from the samples donors the third Affiliated Hospital of Sun Yat-Sen University and the first Affiliated Hospital of Jinan University, indicating that the authenticity of those Proteus strains were proved.
There pairs of primers for conventional PCRs were designed by taking the aptD and tuf genes as objective sequences to amplify the corresponding genes of 3 references Proteus, 66 isolated sample Proteus and 13 non-Proteus strains under the various PCR conditions. Results showed that 3 references Proteus and 66 isolated sample Proteus strains were all positive whereas 13 non-Proteus strains negative, which were exact consistent with the results detected by the conventional methods. However, the newly PCR methods took only 6-8 h, and appeared advantages of rapidity, concise, sensitivity and specificity.
One specific primer specified for SYBR Green I fluorescent real-time PCR, based on atpD gene, were designed for rapid detection of Proteus. The tested results were exact same as the previous conventional PCR methods. However, the time spent for this detection was only 1-2 h, much more quick, specific, sensitive, simple than the conventional methods.
Sequences of 8 bands of aptD genes tested results showed that there are no such sequences of Proteus aptD genes in the existed GenBank. Therefore, the sequences of Proteus aptD genes tested in this study are the first report in the world.
In conclusion, the three conventional PCR methods and one specific fluorescent real-time PCR method are alternative reliable and rapid method applied for the rapid detection and identification of Proteus in food.
引文
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