ABSTRACT
OBJECTIVE: To design specific identification primers based on ribosomal DNA ITS sequences and accurate identification of Dendrobium huoshanensis and its adultments. METHODS: Phylogenetic tree of D. huoshanense and D. officinale and D. henanense by maximum parsimony (MP) and Bayesian inference (BI) were constructed. Specific primers SH-CP9s /SH-CP9a, SHCP25s / SH-CP25a, SH-CP29s /SH-CP29a for PCR identification of D. huoshanensis based on mutation sites on ITS sequences were designed. RESULTS: In the phylogenetic tree, D. huoshanense and D. officinale and D. henanense were clustered into monophyletic group. The specific PCR reaction procedure of D. huoshanense was obtained by investigating the annealing temperature and the amount of DNA template, and different types of PCR and Taq enzymes. In the PCR product, D. huoshanense appearance of the band, and adultments with no band. CONCLUSION: The ITS sequences can be used as DNA Barcoding identification of D. huoshanensis, and identify D. huoshanense and its adultments by the designed primers.
ABSTRACT
BACKGROUND Human trichinellosis is a foodborne parasitic zoonotic disease caused by ingestion of raw or undercooked meat infected with nematode larvae of the genus Trichinella. In the USA, sporadic cases and outbreaks caused by the consumption of wild game meat infected with Trichinella have been reported. The current methods for diagnosis such as serology and microscopy are not specific, may result in false negative results, and cannot differentiate encapsulated Trichinella larvae to species level. The molecular protocols currently available for the differentiation of all encapsulate Trichinella species prevalent in North America have some limitations such as the inability to identify and resolve the presence of several Trichinella species in a single test. OBJECTIVES/METHODS In this study we developed and evaluated a multiplex TaqMan quantitative real-time polymerase chain reaction (qPCR) assay, which can simultaneously detect, identify and differentiate all species of encapsulated Trichinella occurring in North America i.e., T. nativa, T. spiralis, T. murrelli and Trichinella T6, even in cases of multiple infection in a single sample. We investigated two human biopsies and 35 wild animal meat samples considered as having a high likelihood of harboring Trichinella larvae obtained from the United States during 2009-2017. FINDINGS Using the multiplex assay describe here, 22 (59%) samples that tested positive contained Trichinella spp., were identified as: T. nativa (n = 7, including a human biopsy), T. spiralis (n = 9, including a human biopsy), T. murrelli (n = 3), Trichinella T6 (n = 1). Results also included two rare mixed infection cases in bears, a T. nativa/T. spiralis from Alaska and a T. spiralis/Trichinella T6 from California. The species identifications were confirmed using a conventional PCR targeting the rRNA ITS1-ITS2 region, followed by DNA sequencing analysis. The estimated limit of detection (LOD) was approximately seven larvae per gram of meat. MAIN CONCLUSIONS Differentiation of Trichinella spp. is needed to improve efforts on identification of case, optimize food safety control and better understand the geographic distribution of Trichinella species. The Trichinella qPCR multiplex proved to be a robust, easy to perform assay and is presented as an improved technique for identification of all known encapsulated species occurring in North America continent.
Subject(s)
Humans , Trichinella , Microscopy/methods , Polymerase Chain Reaction , Real-Time Polymerase Chain ReactionABSTRACT
OBJECTIVE: To establish a practical, convenient and accurate method of DNA molecular marker for the identification of Testudinis Carapax et Plastrum. METHODS: Phenol extraction method and salting-out method were used to extract mtDNA from Testudinis Carapax et Plastrum and its adulterants. Cyt b mtDNA sequences of Chinemys reevesii and other turtle species were downloaded from GenBank. Species specific PCR primers were designed according to the differential DNA fragments of Cyt b genes, and Cyt b gene segment was amplified by PCR technique and sequenced. RESULTS: The complete 16.6 kb mtDNA was extracted from all samples by the two extraction methods. The specific primers could only amplify the Cyt b gene sequence of Chinemys reevesii, and its fragment was about 319 bp. The result of sequencing indicated that the homologous similarity was 100% with Chinemys reevesii. CONCLUSION: Salting-out method is suitable for the DNA extraction from Testudinis Carapax et Plastrum. The primers are specific to Chinemys reevesii and can be used to distinguish Testudinis Carapax et Plastrum from its adulterants. Copyright 2012 by the Chinese Pharmaceutical Association.