| Objective:To understand the poisoning characteristics of toxic plants in China and to clarify the direction of poisoning control efforts.To evaluate the feasibility of DNA barcode technology for clinical aid diagnosis and screen the most suitable plant sequences.Preliminary construction of a proprietary database of toxic plants to provide data support for rapid traceability of plant poisoning.Methods:1.The Chinese hospital knowledge database(CHKD)and Wanfang database were searched to collect the published literature on the incident of plant poisoning cases in China from 2012 to 2021.Retrospective descriptive epidemiological analysis was performed using Epidata and SPSS software.2.A total of 83 plants were collected between June and December 2020 in Shandong,Zhejiang,Guangxi and Jiangsu Province.The species were initially identified by morphological identification.Thirteen toxic plants were selected and digested by simulated gastric fluid(SGF)for different times(2 h,4 h and 6 h).For all untreated and SGF-treated samples,the target genomes were extracted using the kit method,and the rbcL,trnH-psbA,matK and ITS sequences were amplified by PCR and sequenced in both directions using universal primers.The clinical suitability of each sequence was assessed by comparing the amplification and sequencing success of the samples.3.The measured rbcL,trnH-psbA and ITS sequences were spliced and combined,and all single and combined barcodes were analyzed using the basic local alignment search tool method(BLAST),Barcoding gap analysis,the pair-wise genetic method(PWG)and the neighbor joining tree construction method(NJ tree).The viability of DNA barcoding for gastric contents sample identification was explored by comparing the BLAST results of the samples from the pre and post SGF treatment.The species identification ability of the candidate sequences was evaluated by the sequence characteristics,intra-and interspecific genetic variation,Barcoding gap and phylogenetic tree.The newly measured sequences were also constructed into a proprietary DNA barcode database using database tools.Results:1.Results of literature analysis: a total of 297 literature papers were included,reporting 4331 cases of poisoning and 111 cases of death.The regional distribution of plant poisoning in China is varied.The number of cases in the top 3 areas of poisoning is Yunnan(1096 cases,25.31%),Guangdong(413 cases,9.54%)and Guangxi provinces(357 cases,8.24%).Toxic species are relatively concentrated,most of the poisoning species originated from Ranunculaceae(1856 cases,42.85%)and Leguminosae(1272cases,29.37%).The causes of poisoning can be broadly classified as accidental ingestion or accidental exposure,improper processing or consumption,abuse and suicide,with a high proportion of abuse,including abuse of medicinal alcohol(17.16%)and abuse of herbal medicines(17.39%).2.Amplification and sequencing results: The amplification success rate of matK sequences was low(45.78%)while the amplification success rate of the rest sequences were over 95%.rbcL sequences had the highest sequencing success rate(100%),followed by ITS sequences(74.07%)and trnH-psbA sequences(60.76%).Finally,220 new sequences were obtained.3.SGF results: matK sequences were susceptible to SGF,and most samples showed amplification or sequencing failure after 6 h of treatment.rbcL and trnH-psbA sequences were not affected by SGF,and the amplification and sequencing of all samples were the same as untreated.ITS sequences showed anomalies,some samples showed amplification failure when untreated,but was successfully amplified after SGF treatment.4.DNA barcoding results: The BLAST method showed high identification success for all three sequences at the genus level(trnH-psbA: 98.33%,ITS: 95.83%,rbcL:95.12%),while at the species level ITS and trnH-psbA sequences had higher identification success(83.33%),followed by rbcL sequences(51.22%).And the BLSAT comparison results of all the samples treated with SGF were the same as untreated.All three sequences showed higher interspecies genetic variation than intra-species.The ITS sequences showed a significant advantage over the remaining two sequences in Barcoding gap analysis.The PWG distance method showed that the species identification ability was better for ITS and rbcL sequences(100%)and worse for trnH-psbA sequences(78.28%).The phylogenetic tree showed that the species based on ITS sequences presented the highest percentage of monophyly(100%),followed by rbcL sequences(87.10%)and trnH-psbA sequences(65.22%).Species identification success increased but not significantly when barcodes were combined,with the highest resolution for the ITS +rbcL combination(PWG: 100%,NJ tree: 100%).Local databases were successfully constructed and test samples were correctly identified at the species level.Conclusion:1.Plant poisoning in China has typical regional characteristics,the poison presents aggregation,poisonous plants from the Ranunculaceae and Leguminosae as the main source of poisoning.The causes of poisoning are complex and diverse,with abuse as the major poison-causing factor.2.DNA barcoding can rapidly identify toxic plants and has great potential for clinical application.In this study,the single barcode rbcL sequence is suggested as the barcode of choice for clinical plant poisoning identification,and trnH-psbA could be used as an auxiliary sequence to identify near-derived species when necessary. |
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