鸡白痢沙门氏菌常规和实时荧光定量PCR检测方法的建立
发布时间：2019-06-25 07:45
【摘要】：鸡白痢是由鸡白痢沙门氏菌引起的危害养禽业的细菌病之一,严重影响着我国禽养殖业的健康发展,并且被列为国家规定净化的细菌病。目前,最有效的防控措施是加强对鸡群的检疫、及时淘汰感染鸡只,逐步达到净化鸡群的目的。因此,建立一种快速、准确的检测方法对于鸡白痢的净化工作有着重要意义。本研究中,通过对Gen Bank登录的菌株号为ATCC 9120的鸡白痢沙门氏菌全基因组进行全面生物信息学分析,与NCBI数据库中其他菌属的基因组比较,筛选出了鸡白痢沙门氏菌基因组中的一段基因号为SEEP9120_017695的保守序列,该基因为鸡白痢沙门氏菌的特有基因,因此确定其为检测鸡白痢沙门氏菌的靶基因。根据选出的靶点基因设计了11对候选引物,使用33株沙门氏菌和11株非沙门氏菌以PCR方法分别验证各对引物的特异性,最终筛选出了一对特异性最好且扩增条带大小(219 bp)适合进行荧光定量PCR的引物,经PCR条件优化后建立了一种常规PCR检测方法。对所建立方法的灵敏度进行检验,其检测鸡白痢沙门氏菌基因组时,灵敏度可达2.13 pg/μL;检测细菌纯培养物时灵敏度为2.1×104 cfu/mL。以该方法对人工污染鸡白痢沙门氏菌的鸡粪、鸡蛋和鸡肉进行检测,对于轻度污染(13 cfu/10g样品)的样品,只需要对样品增菌培养6~8小时便可检出;增菌培养2 h便可检测严重污染(1.3×107 cfu/10 g样品)的样品。但是在检测鸡蛋样品时需适当增加1~2小时的增菌时间便可有效检出阳性样品。根据常规PCR方法的反应条件,建立了SYBR荧光定量PCR方法。依据特异性评价实验结果中44个菌株的扩增曲线和熔解曲线,分析可知该方法检测鸡白痢沙门氏菌时的特异性好。建立的SYBR荧光定量PCR方法检测鸡白痢沙门氏菌基因组的灵敏度为34 fg/μL。使用鸡白痢沙门氏菌分别污染鸡粪、鸡蛋、鸡肉,建立人工污染样品,同时使用本研究建立的SYBR荧光定量PCR方法和标准细菌分离鉴定的方法对污染样品进行检测,评价本方法的检测效果。当鸡白痢沙门氏菌的初始污染量低至2 cfu/10 g鸡粪或鸡肉时,经过6 h对样品的振荡培养,荧光定量PCR方法阳性检出率为24/27(88.9%),与标准方法检测的符合率为100%;当以2 cfu/10 mL的初始接种量污染鸡蛋时,6 h后荧光定量PCR方法的阳性检出率为21/27(77.8%),与标准方法检测的符合率为91.7%。本研究筛选到一个鸡白痢沙门氏菌特有的基因作为检测用的靶基因,并以此建立了常规PCR和实时荧光定量PCR检测方法,两种方法在实际检测中各有优势,常规PCR检测方法简便、经济,而荧光定量PCR检测方法具有灵敏度更高、检测更准确的特点,实际检测过程中可以考虑实际情况对两种方法进行选择。本研究为家禽养殖业提供了快速诊断鸡白痢的技术手段,也为实验室今后对鸡白痢沙门氏菌的鉴别检测提供了研究基础。
[Abstract]:Chicken white dysentery is one of the bacterial diseases caused by Salmonella pullorum, which seriously affects the healthy development of poultry breeding in China and is listed as a bacterial disease purified by the state. At present, the most effective prevention and control measures are to strengthen the quarantine of chickens, eliminate infected chickens in time, and gradually achieve the purpose of purifying chickens. Therefore, it is of great significance to establish a rapid and accurate detection method for the purification of chicken white dysentery. In this study, the whole genome of Salmonella pullorum with Gen Bank registration strain number ATCC 9120 was analyzed by comprehensive bioinformatics analysis. Compared with the genomes of other bacteria in NCBI database, a conserved sequence of gene number SEEP9120_017695 in the genome of Salmonella pullorum was screened out, which was a specific gene of Salmonella pullorum, so it was identified as the target gene for the detection of Salmonella pullorum. According to the selected target genes, 11 pairs of candidate primers were designed. 33 strains of Salmonella and 11 strains of non-Salmonella were used to verify the specificity of each pair of primers by PCR. Finally, a pair of primers with the best specificity and amplification band size (219 bp) suitable for fluorescence quantitative PCR were selected. A conventional PCR detection method was established after the optimization of PCR conditions. The sensitivity of the established method was 2.13 pg/ 渭 L for the detection of Salmonella pullorum genome and 2.1 脳 10 ~ 4 cfu/mL. for the detection of pure bacterial culture. The method was used to detect the chicken dung, eggs and chicken of artificially contaminated Salmonella pullorum. For the lightly contaminated samples (13 cfu/10g samples), it was only necessary to culture the samples for 6 to 8 hours, and the samples with severe contamination (1.3 脳 10 7 cfu/10g samples) could be detected after 2 hours of culture. However, the positive samples can be detected effectively by adding 1 to 2 hours of bacteria in the detection of egg samples. According to the reaction conditions of conventional PCR method, SYBR fluorescence quantitative PCR method was established. According to the amplification curve and melting curve of 44 strains in the specific evaluation experiment, it was found that the method had good specificity in the detection of Salmonella pullorum. The sensitivity of the established SYBR fluorescence quantitative PCR method for the detection of Salmonella pullorum genome was 34 fg/ 渭 L. Salmonella pullorum was used to pollute chicken manure, eggs and chicken respectively, and artificial contaminated samples were established. SYBR fluorescence quantitative PCR method and standard bacterial isolation and identification method were used to detect the contaminated samples, and the detection effect of this method was evaluated. When the initial contamination of Salmonella pullorum was as low as 2 cfu/10 g chicken manure or chicken, after 6 hours oscillatory culture of the samples, the positive rate of fluorescence quantitative PCR was 24 鈮，
本文编号：2505517
[Abstract]:Chicken white dysentery is one of the bacterial diseases caused by Salmonella pullorum, which seriously affects the healthy development of poultry breeding in China and is listed as a bacterial disease purified by the state. At present, the most effective prevention and control measures are to strengthen the quarantine of chickens, eliminate infected chickens in time, and gradually achieve the purpose of purifying chickens. Therefore, it is of great significance to establish a rapid and accurate detection method for the purification of chicken white dysentery. In this study, the whole genome of Salmonella pullorum with Gen Bank registration strain number ATCC 9120 was analyzed by comprehensive bioinformatics analysis. Compared with the genomes of other bacteria in NCBI database, a conserved sequence of gene number SEEP9120_017695 in the genome of Salmonella pullorum was screened out, which was a specific gene of Salmonella pullorum, so it was identified as the target gene for the detection of Salmonella pullorum. According to the selected target genes, 11 pairs of candidate primers were designed. 33 strains of Salmonella and 11 strains of non-Salmonella were used to verify the specificity of each pair of primers by PCR. Finally, a pair of primers with the best specificity and amplification band size (219 bp) suitable for fluorescence quantitative PCR were selected. A conventional PCR detection method was established after the optimization of PCR conditions. The sensitivity of the established method was 2.13 pg/ 渭 L for the detection of Salmonella pullorum genome and 2.1 脳 10 ~ 4 cfu/mL. for the detection of pure bacterial culture. The method was used to detect the chicken dung, eggs and chicken of artificially contaminated Salmonella pullorum. For the lightly contaminated samples (13 cfu/10g samples), it was only necessary to culture the samples for 6 to 8 hours, and the samples with severe contamination (1.3 脳 10 7 cfu/10g samples) could be detected after 2 hours of culture. However, the positive samples can be detected effectively by adding 1 to 2 hours of bacteria in the detection of egg samples. According to the reaction conditions of conventional PCR method, SYBR fluorescence quantitative PCR method was established. According to the amplification curve and melting curve of 44 strains in the specific evaluation experiment, it was found that the method had good specificity in the detection of Salmonella pullorum. The sensitivity of the established SYBR fluorescence quantitative PCR method for the detection of Salmonella pullorum genome was 34 fg/ 渭 L. Salmonella pullorum was used to pollute chicken manure, eggs and chicken respectively, and artificial contaminated samples were established. SYBR fluorescence quantitative PCR method and standard bacterial isolation and identification method were used to detect the contaminated samples, and the detection effect of this method was evaluated. When the initial contamination of Salmonella pullorum was as low as 2 cfu/10 g chicken manure or chicken, after 6 hours oscillatory culture of the samples, the positive rate of fluorescence quantitative PCR was 24 鈮，
本文编号：2505517
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