Isothermal Amplification Detection For Lethal Amanitas And The Diversity Of Toxin Genes In Amanita Mushrooms

In recent years,poisoning cases caused by mistaken eating wild mushroom have occured frequently in China,which has became the main factor of food poisoning to death in our country.Meanwhile,lethal Amanita species are responsible for more than 80% of mushroom poisoning to death in our country.Early identification of poisonous mushroom species in poisoning is very important for the diagnosis and treatment of poisoned patients.However,due to the following reasons:(1)the diversity of Amanita species and the incompleteness of samples in the poisoning incident,(2)morphological identification needs professional taxonomists,(3)the current molecular identification requires PCR instrument and DNA sequencing,which takes a very long time,it is difficult to meet the demand for the primary disease control centers and medical institutions to rapid identify toxic source in mushroom posioning.In addition,the transcriptome information of amanitin-containing mushrooms such as lethal Amanita species is insufficient and the related study on toxin gene family is becoming the research hotspot.There are more new cyclic peptide genes and corresponding encoded peptides to be discovered.In this paper,establishment of the isothermal amplification detection methods for lethal amanitas,de novo transcriptome sequencing of lethal Amanita,Galerina and Lepiota speices,diversity of Amanita toxin genes and the prolyl oligopeptidase genes(POP),etc.were studied.The main results were as follows:1.Based on the loop-mediated isothermal amplification technique(LAMP),the internal transcribed spacer(ITS)used as the target,aimed at Amanita bisporigera,A.phalloides in European and America,and A.exitialis,A.fuliginea in China,etc.,ten sets of specific LAMP primers were designed for species-specific detection methods and a set of universal LAMP primers were designed for detecting all lethal amanitas.The LAMP reaction system and conditions(magnesium ion concentration,reaction temperature,time,etc.)were optimized.The amplification product was detected by HNB staining method,and the specificity and sensitivity of the primers were evaluated.The results showed that the reaction results could be obtained by isothermal amplification at 62 °C for 1 h.The DNA detection limit of LAMP was 10 pg.The LAMP-based assay was able to discriminate intro-clade lethal Amanita species but was not able to discriminate intraclade species well.The universal primers could specifically detect lethal amanitas and effectively distinguish between lethal amanitas and non-lethal amanitas.2.Based on the hyperbranched rolling circle amplification technology(HRCA),with ITS as the target sequence,ten specific padlock probes(PLPs)were designed for each of ten lethal amanitas,and then the corresponding species-specific detection methods were established.The universal PLP and the universal PCR primers were designed based on ?-amanitin gene(?-AMA)to establish universal detection methods for lethal amanitas.The HRCA ligation,amplification system and conditions were optimized(PLP concentration,ligation time,etc.),and the amplification product was detected by SYBR Green I staining.The specificity and sensitivity of the probes and the PCR primers were evaluated.The results showed that the reaction results could be obtained by ligation at 60 °C or 65 °C for 1 h,and then amplification at 62 °C for 1 h.Each specific probe could specifically identify the target Amanita speices without cross-reactivity.The universal probe and PCR primers could specifically detect lethal amanitas and distinguish them from non-lethal amanitas and amanitin-containing Galerina and Lepiota speices.The HRCA detection limits were 1 pg Amanita genomic DNA,100 copies of ?-AMA recombinant plasmids and 0.2% mushroom mixture contamination,indicating that the sensitivity of HRCA was 10 times higher than LAMP,100 times higher than conventional PCR.3.Through transcriptome data analysis,PCR amplification using the degenerate primers or the specific primers,MSDIN and POP gene sequences were obtained from 10 Amanita,two Galerina and a Lepiota speices.The results were as follows: 151 MSDIN genes encoded 98 cyclic peptides including ?-amanitin(?-AMA),?-amanitin(?-AMA),phallacidin(PHA)and phalloidin(PHD)and 94 unknown cyclic peptides;Galerina and Lepiota species only contained the MSDIN gene encoding ?-AMA;a total of 10 POPA genes and 9 POPB genes were obtained,and the lethal Amanita,Galerina and Lepiota species contained two types of POP gene,POPA and POPB,while A.oberwinklerana,a Amanita speices containing no amanitin,only contained POPA.4.Structure analysis of ?-AMA and POP showed that: Amanita,Galerina and Lepiota ?-AMA precursor peptides started with MSDIN,MFDTN and MDAN respectively;Amanita and Galerina ?-AMA contained three introns,while Lepiota ?-AMA contained two or three introns;POPA contained 18 introns and POPB contained 17 introns.5.Phylogenetic trees generated from ML analysis based on Amanita toxin genes and POP genes showed that Galerina and Lepiota ?-AMA,Amanita ?-,?-AMA,PHA,PHD were clustered into four clades;Amanita POPA,Galerina POPA,Lepiota POPA,Amanita,Galerina and Lepiota POPB were clustered into four clades.