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Identification of Bupleurum (Apiaceae) seeds by allele-specific PCR based on ITS sequences.

ABSTRACT: The traditional Chinese medicine Bupleuri radix (chaihu) is the dried roots of Bupleurum chinense and Bupleurum scorzonerifolium and many adulterants exist because of the differences in traditional understanding, medication habits and seed resources. Therefore, rapid and accurate identification methods for Bupleurum (Apiaceae) seeds are required. We analyzed the internal transcribed spacer (ITS) sequences of five common Bupleurum species to detect variations in them, including B. chinense, B. scorzonerifolium, B. marginatum var. stenophyllum, B. falcatum and B. smithii var.parvifolium. Based on single nucleotide polymorphisms (SNPs) in the ITS region, we designed five specific primer pairs and used these primers in an allele-specific PCR technique to establish a robust molecular identification method. The neighbor-joining (NJ) tree of ITS sequences showed that five Bupleurum species formed their own monophyly. Five specific primer pairs were designed and integrated into a specific PCR master mix and cycling conditions. The primer pair of BCF/R8 for B. chinense could amplify a specific identification band of 429 bp and the minimum detection limit of the 5 g mixture was about 5%; BSF/R11 for B. scorzonerifolium could amplify a specific 464 bp band and the minimum detection limit was about 5%; BMSF/R1 for B. marginatum var. stenophyllum could amplify a specific 344 bp band and the minimum detection limit was about 1%; BFF/R7 for B. falcatum could amplify a specific 137 bp band and the minimum detection limit was about 1%; BSPF/R1 for B. smithii var. parvifolium could amplify a specific 390 bp band. Subsequent analysis proved the reliable accuracy and good practicability of the five specific identification primers, indicating that the allele-specific primer PCR identification method can quickly identify Bupleurum seeds. The method elaborated in this study has the advantages of simple operation, good accuracy and high efficiency.

SUBMITTER: Qi W

PROVIDER: S-EPMC7210332 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

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Identification of Bupleurum (Apiaceae) seeds by allele-specific PCR based on ITS sequences.

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