雞白痢沙門氏菌常規(guī)和實時熒光定量PCR檢測方法的建立
發(fā)布時間：2019-06-25 07:45
【摘要】：雞白痢是由雞白痢沙門氏菌引起的危害養(yǎng)禽業(yè)的細菌病之一,嚴重影響著我國禽養(yǎng)殖業(yè)的健康發(fā)展,并且被列為國家規(guī)定凈化的細菌病。目前,最有效的防控措施是加強對雞群的檢疫、及時淘汰感染雞只,逐步達到凈化雞群的目的。因此,建立一種快速、準確的檢測方法對于雞白痢的凈化工作有著重要意義。本研究中,通過對Gen Bank登錄的菌株號為ATCC 9120的雞白痢沙門氏菌全基因組進行全面生物信息學(xué)分析,與NCBI數(shù)據(jù)庫中其他菌屬的基因組比較,篩選出了雞白痢沙門氏菌基因組中的一段基因號為SEEP9120_017695的保守序列,該基因為雞白痢沙門氏菌的特有基因,因此確定其為檢測雞白痢沙門氏菌的靶基因。根據(jù)選出的靶點基因設(shè)計了11對候選引物,使用33株沙門氏菌和11株非沙門氏菌以PCR方法分別驗證各對引物的特異性,最終篩選出了一對特異性最好且擴增條帶大小(219 bp)適合進行熒光定量PCR的引物,經(jīng)PCR條件優(yōu)化后建立了一種常規(guī)PCR檢測方法。對所建立方法的靈敏度進行檢驗,其檢測雞白痢沙門氏菌基因組時,靈敏度可達2.13 pg/μL;檢測細菌純培養(yǎng)物時靈敏度為2.1×104 cfu/mL。以該方法對人工污染雞白痢沙門氏菌的雞糞、雞蛋和雞肉進行檢測,對于輕度污染(13 cfu/10g樣品)的樣品,只需要對樣品增菌培養(yǎng)6~8小時便可檢出;增菌培養(yǎng)2 h便可檢測嚴重污染(1.3×107 cfu/10 g樣品)的樣品。但是在檢測雞蛋樣品時需適當(dāng)增加1~2小時的增菌時間便可有效檢出陽性樣品。根據(jù)常規(guī)PCR方法的反應(yīng)條件,建立了SYBR熒光定量PCR方法。依據(jù)特異性評價實驗結(jié)果中44個菌株的擴增曲線和熔解曲線,分析可知該方法檢測雞白痢沙門氏菌時的特異性好。建立的SYBR熒光定量PCR方法檢測雞白痢沙門氏菌基因組的靈敏度為34 fg/μL。使用雞白痢沙門氏菌分別污染雞糞、雞蛋、雞肉,建立人工污染樣品,同時使用本研究建立的SYBR熒光定量PCR方法和標準細菌分離鑒定的方法對污染樣品進行檢測,評價本方法的檢測效果。當(dāng)雞白痢沙門氏菌的初始污染量低至2 cfu/10 g雞糞或雞肉時,經(jīng)過6 h對樣品的振蕩培養(yǎng),熒光定量PCR方法陽性檢出率為24/27(88.9%),與標準方法檢測的符合率為100%;當(dāng)以2 cfu/10 mL的初始接種量污染雞蛋時,6 h后熒光定量PCR方法的陽性檢出率為21/27(77.8%),與標準方法檢測的符合率為91.7%。本研究篩選到一個雞白痢沙門氏菌特有的基因作為檢測用的靶基因,并以此建立了常規(guī)PCR和實時熒光定量PCR檢測方法,兩種方法在實際檢測中各有優(yōu)勢,常規(guī)PCR檢測方法簡便、經(jīng)濟,而熒光定量PCR檢測方法具有靈敏度更高、檢測更準確的特點,實際檢測過程中可以考慮實際情況對兩種方法進行選擇。本研究為家禽養(yǎng)殖業(yè)提供了快速診斷雞白痢的技術(shù)手段,也為實驗室今后對雞白痢沙門氏菌的鑒別檢測提供了研究基礎(chǔ)。
[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，
本文編號：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，
本文編號：2505517
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