Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Establishment of mink heart identification method based on mitochondrial cytochrome b gene and development of its detection kit.

In this study, the mink heart mitochondrial DNA was used as the target gene to design the specific primers of mink heart mtDNA Cytb, the extraction and detection reagents of mink heart DNA were developed using DNA fingerprinting technique, and the specificity, reproducibility, and stability of the reagents were investigated. Molecular cloning and sequencing technique were used to clone the standard substance of mink heart DNA detection, then a DNA fingerprint detection method of mink heart and the quality standard for mink heart were established, and a DNA detection kit of mink heart was developed. The results showed that the structure of DNA extracted from mink heart by self-developed reagents was complete, and both the concentration and purity of DNA were high. A specific amplification band of the original mink samples was found at 337 bp. The sequence of mink heart DNA was consistent with that of mink heart mtDNA specific fingerprint region. The mink heart DNA fingerprint identification method established, in this study, is accurate and reliable, and the procedure of the developed DNA kit is easy, the results obtained using this kit is stable and the method is suitable for popularization and application.

Study of introduction apoptosis of hepatocarcinoma cell SMMC-7721 by anthraquinone extracted from rubiqmnone and molecular mechanism / 中国中药杂志

<p><b>OBJECTIVE</b>To research the effect of anthraquinone extracted from rubiqmnone on growth inhibition, introduction apoptosis and expression of relative gene of bcl-2 of hepatocarcinoma cell SMMC 7721, and provide the effective target of gene therapy.</p><p><b>METHOD</b>The growth inhibition effect was detected by MTF. Morpholgy, DNA agarose gel electrophoresis and flow cytometry were used to observe the cell apoptosis. The effect of anthraquinone on bcl-2mRNA expression was analyzed by RT-PCR.</p><p><b>RESULT</b>Anthraquinone could inhibit the growth of hepatocarcinoma cell SMMC 7721. The typical apoptosis cells were found by inverted microscope and common microscope. DNA agarose gel electrophoresis showed a typical apoptosis strip. G1 period of cell cycle had apoptosis peak of abnormal diploid by PI simple stain, and cell cycle stopped at G1 period. The apoptosis fuction of anthraquinone introdution hepatocarcinoma cell was further certified by Annexin V-FITC/PI. Anthraquinone could decrease the expression of bcl-2mRNA by RT-PCR.</p><p><b>CONCLUSION</b>Anthraquinone can significantly inhibit growth of hepatocarcinoma cell and induce apoptosis. The mocular mechanism may be related to down-regulating the expression of bcl-2 gene.</p>