串珠镰刀菌伏马菌素产毒株聚合酶链反应检测方法的研究  

Study on the polymerase chain reaction methods for the detection of fumonisin-producing strains of Fusarium moniliforme

作　　者：王晓英[1] 刘秀梅[1] 

机构地区：[1]中国疾病预防控制中心营养与食品安全所,北京100050

出　　处：《卫生研究》2003年第3期228-232,共5页Journal of Hygiene Research

基　　金：国家自然科学基金资助项目 (No .39870 675)

摘　　要：以伏马菌素生物合成所必需的多酮肽合成酶基因fum5为基础 ,设计了 3对特异性引物P1 P2、P3 P4和Fum5F2 1 Fum5 1,建立了串珠镰刀菌伏马菌素产毒株PCR检测方法。以 5株ATCC典型串珠镰刀菌及其他菌株为参考 ,其中ATCC 5 2 5 3 9为伏马菌素B1(FB1)产毒株 ,测定了PCR方法的特异性。ATCC 5 2 5 3 9扩增结果阳性 ,该 3对引物分别扩增出 880bp、70 2bp和 10 40bpDNA片段 ,非伏马菌素产毒株ATCC 3 8946、ATCC2 62 63、ATCC 12 763、ATCC 3 80 16及其他阴性对照菌株 (禾谷镰刀菌、梨孢镰刀菌、木贼镰刀菌、三线镰刀菌、黄曲霉、拟青霉和大肠杆菌O15 7)结果阴性 ,证明引物具有很好的特异性。同时以不同稀释度的ATCC 5 2 5 3 9DNA为模板 ,测定了 3对引物PCR的敏感性 ,P1 P2每PCR反应的检出限为 10 0pg ,P3 P4和Fum5F2 1 Fum5R1每PCR反应的检出限均为 10pg ,分别相当于每PCR反应检出 10 4和 10 3个孢子。 3对引物PCR检测国内不同地区分离的 3 2株串珠镰刀菌及交孢变种 ,2 6株为伏马菌素产毒株 ,6株为非伏马菌素产毒株。并对部分产毒株PCR(P3 P4)产物 ,应用内切酶EcoRV和BamHI,进行限制片段长度多态性 (RFLP)分析 ,分别产生 181bp、5 2 1bp 2个和 116bp、2 5 8bp、3 2 8bp 3个DNA片段 ,与文献报道值一致 。Three pairs of PCR primers P1/P2, P3/P4 and Fum5F21/ Fum5R1 specific for Fumonisin-producing were designed, based on the polyketide synthesase gene fum5 of Fusarium moniliforme involved in fumonisin biosynthesis. PCR methods for detecting Fumonisin-producing F. moniliforme strains were developed. The specificity of PCR with 3 pairs of primers was detected with 5 standard F. moniliforme strains from ATCC. 880bp、702bp and 1040bp DNA fragments were amplified with P1/P2, P3/P4 and Fum5F21/ Fum5R1, respectively, by using ATCC 52539 (Fumonisin-producing strain) as DNA template . Negative results were obtained by using ATCC 38946、ATCC 26263、ATCC 12763、 ATCC 38016 (Fumonisin-non-producing strains), and other negative control strains such as F. gramnearum, F. poae, F. equiseti, F. tricinctum, A. flavus and E.coli O157. The sensitivity of PCR with 3 pairs of primers was detected by applying different dilutions of ATCC52539 DNA template. The detection limits for PCR with P1/P2 was 100pg per PCR assay, and with P3/P4 and Fum5F21/ Fum5R1 were all 10pg, equivalently 10 4 and 10 3 spores per PCR assay, respectively. Total 32 strains of F. moniliforme and F. moniliforme var isolated from different regions in China were identified by using PCR with P1/P2, P3/P4 and Fum5F21/ Fum5R1. 26 out of them were identified as Fumonisin-producing strains, and other 6 isolates as Fumonisin-non-producing strains. Restriction fragment length polymorphism (RFLP) analysis for PCR (P3/P4) products of some Fumonisin-producing strains were performed by using restriction endonuclease EcoR V and Bam HI. Two DNA fragments 181bp、521bp and three fragments 116bp、258bp、328bp were produced, respectively, which accorded with reported reference value. Reliability of the results was confirmed. The results indicated that only one strain, ZJ-fm052, had Fumonisin biosynthesis gene, but negative in detecting fumonisin B1 in culture. Further studies are required. It proved that PCR methods are rapid and reliable to detect Fumonisin-producing strain

关 键 词：串珠镰刀菌 伏马菌素产毒株 聚合酶链反应 限制片段长度多态性分析 

分 类 号：R155.5[医药卫生—营养与食品卫生学]