Wang Xiaoying, Liu Xiumei
Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Wei Sheng Yan Jiu. 2003 May;32(3):228-32.
Three pairs of PCR primers P1/P2, P3/P4 and Fum5F21/Fum5R1 specific for Fumonisin-producing were designed, based on the polyketide synthase 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. 880 bp, 702 bp and 1040 bp 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 ATCC 52539 DNA template. The detection limits for PCR with P1/P2 was 100 pg per PCR assay, and with P3/P4 and Fum5F21/Fum5R1 were all 10 pg, 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 181 bp, 521 bp and three fragments 116 bp, 258 bp, 328 bp 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 strains.
基于参与伏马菌素生物合成的串珠镰刀菌聚酮合酶基因fum5，设计了三对用于检测产伏马菌素菌的PCR引物P1/P2、P3/P4和Fum5F21/Fum5R1。建立了检测产伏马菌素串珠镰刀菌菌株的PCR方法。用来自美国典型培养物保藏中心（ATCC）的5株标准串珠镰刀菌菌株检测了这3对引物PCR的特异性。以ATCC 52539（产伏马菌素菌株）为DNA模板，用P1/P2、P3/P4和Fum5F21/Fum5R1分别扩增出880 bp、702 bp和1040 bp的DNA片段。以ATCC 38946、ATCC 26263、ATCC 12763、ATCC 38016（不产伏马菌素菌株）以及其他阴性对照菌株如禾谷镰刀菌、燕麦镰刀菌、木贼镰刀菌、三线镰刀菌、黄曲霉和大肠杆菌O157作为模板时，结果均为阴性。通过对ATCC 5
