Isolation and Identification of Pathogenic Yersinia enterocolitica and Development of a Triple PCR Detection Method
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摘要: 为鉴定导致患病鲫鱼肛门红肿、腹部有出血点症状的病原菌，并建立一种快速检测该病原菌的方法。本研究从患病鲫鱼中分离了病原菌，采用形态学、理化特性分析及16S rDNA序列分析方法鉴定菌株。采用PCR扩增法检测该菌毒力基因，琼脂纸片扩散法检测该菌株的耐药性，致病性能验证试验检测其致病性。针对该菌ail基因、inv基因和intB基因设计了3条特异性引物，通过对反应体系和条件的优化，建立了一种检测该菌的三重PCR方法并初步应用于患病鲫鱼样品的检测中。结果显示，从患病鲫鱼心脏组织中分离了一株小肠结肠炎耶尔森氏菌（Yersinia enterocolitica），命名为fsznc-10。检测到该菌携带ail、ystB、virF、intB毒力基因，该菌对诺氟沙星、庆大霉素等5种抗生素敏感，对鲫鱼具有一定的致病性。三重PCR方法可准确扩增出小肠结肠炎耶尔森氏菌ail、inv和intB三个目的基因，而其他菌株均未扩增出目的基因。该方法检测该菌DNA最低检出量为1.704×10−6 ng/μL，检测患病鱼心脏样品的阳性率约为86.67%，与16S rDNA序列分析方法的检测符合率为100%。本试验建立的三重PCR检测法具有特异性强、敏感性高、操作简单、成本低的优点。这为临床中小肠结肠炎耶尔森氏菌的防控和检测提供参考依据。
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关键词:
- 小肠结肠炎耶尔森氏菌 /
- 毒力基因 /
- 快速检测 /
- 多重PCR
Abstract: This paper aimed to identify the pathogenic bacterium that caused anal swelling and abdominal bleeding of Carassius auratus, and establish a rapid detection method for the pathogenic bacterium. In this study, the pathogenic bacterium was isolated from Carassius auratus, and identified by morphological, physical and chemical characteristics analysis and 16S rDNA sequence analysis. The virulence gene of the bacterium was detected by PCR method. The drug sensitivity of isolated strain was detected by disk agar diffusion method and its pathogenicity was discussed by artificial infection. Three specific primers of ail gene, inv gene and intB gene were designed for the isolated strain. A triple PCR method for detecting the isolated strain was established and this method was used to test samples of sick Carassius auratus. The result showed that a Yersinia enterocolitica strain named fsznc-10 was isolated from the heart of diseased Carassius auratus. Yersinia enterocolitica fsznc-10 had virulence genes of ail, ystB, virF and intB. Yersinia enterocolitica fsznc-10 was nonresistant to five antibiotics such as norfloxacin and gentamicin, and it had certain pathogenicity to Carassius auratus. Triple PCR method could accurately amplify three target genes of ail, inv and intB of Yersinia enterocolitica, while other strains had not amplified the target genes. The minimum template concentration for the detection of Yersinia enterocolitica was 1.704 × 10−6 ng/μL. The positive rate of the triple PCR method for detecting heart samples of sick fish was about 86.67%, and its detection coincidence rate with the 16S rDNA sequence analysis method was 100%. The triple PCR detection method has the advantages of strong specificity, high sensitivity, simple operation and low cost. The present paper research provides scientific data reference for the prevention, control and detection of Yersinia enterocolitica in clinical practice.-
Key words:
- Yersinia enterocolitica /
- virulence gene /
- rapid detection /
- multiplex PCR
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图 1 分离菌16S rDNA基因PCR扩增结果
Figure 1. PCR amplification results of 16S rDNA gene of the isolate strain
注：M: DL2000DNA Marker; 1: fsznc-10
图 2 菌株fsznc-10 16S rDNA基因序列的系统发育树
Figure 2. Phylogenetic tree of strain fsznc-10 16S rDNA gene sequence
图 3 毒力基因的PCR扩增结果
注：M: DL2000; 1: ail; 2: ystB; 3: virF; 4: yadA; 5: intB。
Figure 3. PCR amplification of virulence gene
图 4 单一PCR与多重PCR扩增结果
注：M: DL2000DNA Marker; 1: ail; 2: inv; 3: intB;4: 三重PCR结果。
Figure 4. Results of single PCR and multiplex PCR amplifications
图 5 三重PCR的特异性检测
注：M：DL2000DNA Marker；1～15：fsznc-10、假结核耶尔森氏菌、维氏气单胞菌、哈维氏弧菌、门多萨假单胞菌、霍乱弧菌、嗜水气单胞菌、弗劳地枸橼酸杆菌、中间气单胞菌、粪肠球菌、大肠埃希氏菌、金黄色葡萄球菌、类志贺邻单胞菌、霍氏肠杆菌、无丙二酸枸橼酸杆菌；16：阴性对照。
Figure 5. Specificity detection of triple PCR
图 6 三重PCR的敏感性试验结果
注：M：DL2000DNA Marker；1～10：菌株fsznc-10稀释100～109倍；11：阴性对照。
Figure 6. Sensitivity test results of triple PCR
图 7 临床样品三重PCR检测结果
注：M：DL2000DNA Marker；1～15：临床样品；16：阴性对照。
Figure 7. Triple PCR results of clinical samples
表 1 小肠结肠炎耶尔森氏菌毒力基因引物序列
Table 1. Primers of the virulence genes in the Y. enterocolitic strains
引物 序列（5'→3'） 预测长度（bp） GenBank登录号 退火温度（℃） ail Forward taa tgt gta cgc tgc gag 351 M29945 50.0 Reverse gac gtc tta ctt gca ctg ystB Forward gta cat tag gcc aag aga cg 200 GU229276 60.3 Reverse gca aca tac ctc aca aca cc virF Forward ggc aga aca gca gtc aga cata 561 NC004564. 45.0 Reverse ggt gag cat aga gaa tac gtc g yadA Forward ctt cag ata ctg gtg tcg ctg t 800 NC004564 58.5 Reverse atg cct gac tag agc gat atc c intB Forward tgc gcc atg cgg tcc atc 714 NC008800 50.0 Reverse ggt gca taa gat tct cgg Inv* Forward ctg tgg gga gac tgg gga agt ttg g 520 CAA88188 50.0 Reverse gaa ctg ctt gaa tcc ctg aaa acc g 注：*仅应用于菌株fsznc-10的三重PCR检测。 
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表 2 药敏实验结果
Table 2. Results of antibiotic sensitivity test
抗菌药物 结果 抗菌药物 结果 氨苄青霉素 R 诺氟沙星 S 庆大霉素 S 多粘菌素 S 头孢曲松 I 呋喃唑酮 R 阿米卡星 S 链霉素 S 复方新诺明 I 头孢噻吩 R 注：R：耐药；I：中度敏感；S：敏感。
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