Dataset Information

Detection of Highly Poisonous Nerium oleander Using Quantitative Real-Time PCR with Specific Primers.

ABSTRACT: Nerium oleander is one of the most poisonous plants, and its accidental ingestion has frequently occurred in humans and livestock. It is vital to develop a rapid and accurate identification method for the timely rescue of oleander-poisoned patients and the investigation of poisoning cases. In this study, a specific and highly sensitive quantitative real-time PCR (qPCR)-based method was developed to identify oleander in mixture systems and simulated forensic specimens (SFS). First, a new pair of oleander-specific primers, JZT-BF/BR, was designed and validated. Then, a qPCR method was developed using the primers, and its detective sensitivity was examined. The results showed that JZT-BF/BR could specifically identify oleander in forage and food mixtures, and qPCR was capable of accurate authentication even at a low DNA concentration of 0.001 ng/μL. This method was further applied to the analysis of SFS containing different ratios of N. oleander. The method was confirmed to be applicable to digested samples, and the detection limit reached 0.1% (w/w) oleander in mixture systems. Thus, this study undoubtedly provides strong support for the detection of highly toxic oleander and the diagnosis of food poisoning in humans and animals.

SUBMITTER: Bai X

PROVIDER: S-EPMC9696062 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Detection of Highly Poisonous <i>Nerium oleander</i> Using Quantitative Real-Time PCR with Specific Primers.

Bai Xuanjiao X Wang Gang G Ren Ying Y Han Jianping J

Toxins 20221110 11

<i>Nerium oleander</i> is one of the most poisonous plants, and its accidental ingestion has frequently occurred in humans and livestock. It is vital to develop a rapid and accurate identification method for the timely rescue of oleander-poisoned patients and the investigation of poisoning cases. In this study, a specific and highly sensitive quantitative real-time PCR (qPCR)-based method was developed to identify oleander in mixture systems and simulated forensic specimens (SFS). First, a new p ...[more]

PMID: 36356026

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Project description:In this study, phytochemical analysis and toxicity profile of leaf and flower extracts of Nerium oleander L. species collected from Giresun province (Turkey) were investigated. In phytochemical analyzes, the cardiac glycoside, alkaloid, saponin and tannin contents of the extracts were analyzed qualitatively and quantitatively. The physiological effects of extracts were determined by examining root elongation, weight gain and germination rates. Biochemical effects were determined by measuring the levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), which are indicators of oxidative stress. Cytotoxic and genotoxic effects were investigated by mitotic index (MI), micronucleus (MN) and chromosomal abnormality (CA) tests. N. oleander leaf and flower extract applications caused significant decreases in the physiological parameters of Allium bulbs. SOD and CAT activity in root tip cells increased significantly after the application of leaf extract compared to the control group. Similar changes were observed in the application of flower extract, but these increases were found to be at a lower level compared to the increases induced by the leaf extract. An increase in MDA levels and a decrease in GSH levels were observed in groups treated with leaf and flower extracts. These changes show that the extracts cause deterioration in antioxidant/oxidant balance. It was determined that the extracts, which caused a decrease in MI rates and an increase in MN and CAs frequencies, showed the most prominent cytotoxic and genotoxic effects at 250 μg/mL doses. These toxic effects were associated with the phytochemical content of the extracts, and it was thought that cardiac glycosides and alkaloids, whose presence were detected in qualitative and quantitative analyzes, may play an important role in toxicity. Studies investigating the therapeutic properties of plants as well as their toxic effects are insufficient, which leads to the fact that plants exhibiting potential toxicity are not well known. Therefore, this study will lead many studies on the toxicity profile of the phytochemical contents of plants. Therefore, this study will draw attention to the investigation of the toxicity profile and phytochemical contents of plants and will lead to similar studies.

Project description:BackgroundCulture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora.ResultsDesign of specific primers and probe was achieved based on comparison of 108 published bifidobacterial 16S rDNA sequences with the recently published sequence of the human faecal isolate B. thermophilum RBL67. Specificity of the primer was tested in silico by similarity search against the sequence database and confirmed experimentally by PCR amplification on 17 Bifidobacterium strains, representing 12 different species, and two Lactobacillus strains. The qPCR assay developed was linear for B. thermophilum RBL67 DNA quantities ranging from 0.02 ng/microl to 200 ng/microl and showed a detection limit of 10(5) cells per gram faeces. The application of this new qPCR assay allowed to detect the presence of B. thermophilum in one sample from a 6-month old breast-fed baby among 17 human faecal samples tested. Additionally, the specific qPCR primers in combination with selective plating experiments led to the isolation of F9K9, a faecal isolate from a 4-month old breast-fed baby. The 16S rDNA sequence of this isolate is 99.93% similar to that of B. thermophilum RBL67 and confirmed the applicability of the new qPCR assay in faecal samples.ConclusionA new B. thermophilum-specific qPCR assay was developed based on species-specific target nucleotides in the 16S rDNA. It can be used to further characterize the composition of the bifidobacterial community in the human gastrointestinal tract. Until recently, B. thermophilum was considered as a species of animal origin, but here we confirm with the application of this new PCR assay the presence of B. thermophilum strains in the human gut.

Dataset Information

Detection of Highly Poisonous Nerium oleander Using Quantitative Real-Time PCR with Specific Primers.

Publications

Detection of Highly Poisonous <i>Nerium oleander</i> Using Quantitative Real-Time PCR with Specific Primers.

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