| Mushroom poisoning is one of the public health emergencies with the highest mortality rate and the largest number of deaths in food poisoning in China.The rapid and accurate identification of poisonous mushrooms is of vital importance to the prevention of mushroom poisoning and the accurate and effective treatment after poisoning.Among the deaths caused by mushroom poisoning in China,in addition to the accidental eating of the lethal Amanitas or the species of Lepiota genus and Galerina genus,it was mainly caused by the accidental eating of Russula subnigricans.R.subnigricans has become one of the deadly poisonous mushrooms.Meanwhile,in recent years,among the poisoning cases caused by poisonous mushroom which lead to gastroenteritis symptoms in China,the number of poisoning cases caused by R.japonica ranks second for consecutive years.Due to the variety of species of the Russula genus,it is difficult to distinguish the poisonous species from some edible species for their similar morphology,which brings difficulties to species identification.However,conventional DNA barcoding identification requires species sequencing,which takes a long time.Therefore,in order to detect and identify the toxic mushrooms of the genus Russula more quickly and accurately,in this paper,we use the internal transcribed spacer(ITS)as the target to design primers for isothermal amplification molecular detection of Russula subnigricans and R.japonica.The establishment of the method has been studied,and the main results were as follows:1.The reaction system of loop-mediated isothermal amplification(LAMP)technology was established and optimized.The optimal reaction system for LAMP was: the final concentration of magnesium ion was 6m M,the final concentration of hydroxynaphthol blue(HNB)was 180 μM,and the final concentration of Bst DNA polymerase was 0.24 U/μL.The reaction system could have ideal amplification effect at 62°C for 45-60 min,the visualization system based on HNB staining is sky blue.2.Based on the LAMP technology,the LAMP specific primers were designed for R.subnigricans and R.japonica to realize the species-specific detection of R.subnigricans and R.japonica.The results showd that LAMP technology could realize the specific distinction between different subgenus species within the genus Russula quickly and effectively,and could also distinguish species within the same section(R.section Nigricantinae)that were closely related to each other.Based on the PCR reaction principle,we designed the species-specific PCR primers for R.subnigricans.The results showed that traditional PCR could not effectively distinguish the species in the section,and the LAMP reaction was more specific than traditional PCR.The DNA molecular detection limit of the LAMP technique based on HNB staining method was 10 pg,and the agarose gel electrophoresis method could reach 1 pg;the DNA molecular detection limit of PCR was 100 pg.Therefore,the LAMP technology could sensitively realize the specific detection of R.subnigricans and R.japonica.3.Based on the hyper-branched rolling circle amplification(HRCA)technology,a specific padlock probe(PLP)was designed for R.japonica,which realized the species-specific detection of R.japonica.The visualization system based on SYBR GreenⅠ staining is yellow-green. |
