Development Of Gene Chip Based Method For Detection Of Colletotrichum Fungi

Colletotrichum causes anthracnose on plants which causing substantial economic losses to crop production.The C.kahawae is an quarantine pathogen in China.The traditional method for detecting the Colletotrichum is based on the morphological characteristics with the long identification time.It is necessary to establish a method for rapidly detecting the Colletotrichum.In this study,in order to establish a routine PCR rapid detection system for the Colletotrichum witch the ITS gene sequence of the Colletotrichum was analyzed and the conventional PCR-specific primers were designed.Designed a gene chip detection probe for the Colletotrichum,C.kahawa,C.gloeosporioides,C.fructicola and C.destructivum to established a rapid detection system for gene chips which could provide technical support with rapid detection of field diseases and inbound and outbound samples.The results obtained in this study are as follows:The ITS gene of C.olletotrichum and its allied genera were multipling sequence alignments to designed primers ITS-c3/c4 in the specific interval of Colletotrichum.A bright 449 bp band was amplified by routine PCR amplification from 40 different species strains of Colletotrichum.The specificity was verified by 6 strains of the Colletotrichum allied genera and the bands were not amplified which indicating that the designed primers had specificity.The minimum concentration of DNA detectable in the sensitivity test was 2.2 pg/?L.Using the established system to detecte the collected anthracnose samples wihch could amplified a band of about 449 bp.In order to establish a gene chip detection system of the Colletotrichum the detect probes I-P1 and I-P2 were designed for the ITS gene of Colletotrichum.Primers Gapdh-1/R1,GS-k3/k4 and probes G-P1,G-Pkl,G-Pk2 were designed for the GAPDH and GS genes of the C.kahawae.Primers Tub2-1/R1,HIS-g1/g2 and probes T-P1,T-P2,H-P1 were designed for the TUB2 and HIS3 genes of the C.gloeosporioides.Primers Act-1/R1,Gapdh-d1/d2 and probe A-P1,G-Pd1 were designed for the ACT and GAPDH genes of the C.destructivum.Primer SOD-1/f2 and probes S-P2,S-P3,S-P4 were designed for the SOD2 gene of C.fructicola.Four pathogens were amplified by fluorescent-labeled PCR using the designed primers which amplified the bands were consistent with the target size.The hybridization conditions of the gene chip were optimized.When the hybridization time was 60 min and the hybridization temperature was 45? and the SSC concentration of the hybridization solution was 6×SSC that the signal of chip hybridization was the best.In the sensitivity test of gene chip detection for Colletotrichum and C.kahawae and C.gloeosporioides and C.destructivum and C.fructicola the lowest DNA concentration was 0.027pg/?L,5.5 pg/?L,0.27 pg/?L,2.2 pg/?L and 2.9 pg/?L.The sensitivity of gene chip detection was 10-100 times higher than that of ordinary PCR detection which indicate the sensitivity of gene chip detection was higher.The specificity of the designed primers verification showed that 40 strains of Colletotrichum had hybridization signals at the I-P1 probe and there was no hybridization signals at the I-P2 probe which indicate that the I-P1 probe could detect Colletotrichum fungus.Six strains of Colletotrichum's allied genera were detected by gene chip and there was no hybridization signal at the genus probes which indicated that probe I-P1 had specificity and could detect Colletotrichum fungus.The other probes were tested by 40 strains of Colletotrichum and each probe had a hybridization signal only when the corresponding pathogen were detected which indicated that the designed probe had specificity.The plant anthracnose sample was detected which the sample had a hybridization signal at the probe I-P1 with the established gene chip detection method and the results were reliable.In this study we constructed a gene chip method for simultaneous detection of Colletotrichum and C.kahawae and C.gloeosporioides and C.destructivum and C.fructicola.