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Objective To carry out genetic polymorphism analysis of Citrus reticulata Blanco cv. chachiensis Tanaka and its relatives by using SCoT molecular marker method. Methods Five factors of Mg2+, dNTPs, TaqDNA polymerase, primer, and template DNA concentration were used to screen the suitable SCoT-PCR reaction system for C. reticulata and its relatives by the method of orthogonal design. The optimum annealing temperature was screened by gradient temperature, and its polymorphic primers were verified. Results A total of 12 clear and rich bands were finally screened out as the primers of SCoT molecular marker for C. reticulata and its relatives in the optimized PCR reaction system, and the genetic distance and the UPGMA clustering tree of C. reticulata and its relatives were got by NTSYS software analysis. Conclusion The optimized SCoT-PCR reaction system was validated by using three different places of citrus genomic DNA to obtain the polymorphism and the clear amplified bands, which showed that the SCoT molecular marker system of Citrus reticulata is stable and reliable. The results of cluster analysis were able to make a preliminary separation of 13 kinds of materials scientifically and intuitively in molecule level.
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Objective To analyze the variation characteristics of rpoB,katG,inhA,rpsL and embB related genes of Mycobacterium tuberculosis(MTB)in Qinzhou,Guangxi.Methods PCR reverse point hybridization was used to detect 5 common resistance mutants of Mycobacterium tuberculosis in 237 MTB-DNA positive sputum samples.Results Among 237 cases of tuberculosis patients,72 cases(30.38%)were resistant to the four kinds of anti-TB drugs.The resistance mutation rate of rifampin,isoniazid and streptomycin was 2.53%, 13.92%,3.80%.The top 5 gene mutation detection loci of Mycobacterium tuberculosis were-15M,S531L and 43M.Conclusion The main drug-resistant strains are isoniazid resistance,and the mutation of inhA gene were the major one in Qinzhou,Guangxi.
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For rapid identification of Cervus nippon, C. elaphus and their hybridize samples, the specific PCR for mutual authentication of them was established based on the SNPs in COI and SRY sequence. C. nippon, C. elaphus and their hybridize samples were collected from different origins, total DNA of 24 identified samples were extracted, and the COI and SRY gene was seqenced. SNPs in the COI and SRY sequences of the samples were found by Clustul X 2.1 program. Primers for identifying C. nippon and C. elaphus were designed according to the SNP site, two multi-PCR reaction system were established to identify them. In addition, 24 samples which were randomly collected in different herbal medicine market were identified. The band special for C. nippon (232 bp)and band special for C. elaphus (518 bp) based on COI sequence,and the band special for C. nippon (803 bp)and band special for C. elaphus (425 bp) based on SRY sequence, were found using multi-PCR reaction, and three of the twenty-four samples were identified as the hybridize samples. The multi-PCR reaction system could be used to identify C. nippon, C. elaphus and their hybridize samples.
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Objective: For rapid identification of Houttuynia cordata and Gymnotheca chinensis, the specific PCR for mutual authentication of them was established based on the SNPs in matK sequence. Methods: H. cordata and G. chinensis samples from different origins were collected, total DNA of all samples was extracted, and the matK gene was seqenced. SNPs in the matK sequences of all the samples were found by ClustulX 2.1 program. Primers for identifying H. cordata and G. chinens were designed according to the SNP site, and specific PCR method was established to identify them, for rapid detection by addition of SYBR Green I dye. In addition, constructing a multi-PCR reaction system, and then the PCR reaction system was optimized. Results: The band special for H. cordata (185 bp) and band special for G. chinensis (389 bp) were found using specific PCR reaction and multi-PCR reaction, and SYBR Green I dye can be used for rapid detection. Conclusion: The multi-PCR reaction system could be used to identify H. cordata and G. chinensis.
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Objective To establish a simple , fast and economic total DNA extraction method to serve the rapid identification of model mouse genotype in large number of mice .Methods Three methods, i.e.phenol extraction, isopropyl alcohol precipitation and mouse ear boiling methods were used to extract the total DNA from ten C 57-rasmodel mice.The purity and yield of DNA obtained by the three methods were compared , and polymerase chain reaction ( PCR) assay was used to compare the efficacy of the three extraction methods .Results Among the three methods , phenol extraction was the best and isopropyl alcohol precipitation was the poorest in DNA yield .In terms of DNA purity , the phenol extraction was the best and the mouse ear boiling method was the poorest .All the three methods could be used to extract the total DNA from mice serving as template of PCR reaction for the mouse genotype identification .The time consumption of three methods are 12.5 hr ,13 hr and 0.18 hr.Mouse ear boiling method was significantly lower than that of the other two methods ( P <0.01 ) ,.The obtained total DNA can be stored at conventional -20℃for 7 days and 30 days later still can be used as a template for PCR reaction .Conclusions Among the three methods studied , the mouse ear boiling method is simple and with the lowest cost , so it is feasible for total DNA extraction in scaled genotyping experiments .
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Objective: TO establish methods for sensitive, rapid, economical detection of Th1/Th2 Cytokine gene expression. Method: sixpairs of Primers specific for the IL-2, IL-4, IL-10, IL-13, IFNr and B-actin were designed. A Sensitive and rapid method for detection of Th1/Th2cytokine genes by RT-RCR was established. Meanwhile another method: the RNA hybridization with sir cDNA probes were established. Results:The RT-PCR reaction and RNA hybridization was used to test five kinds of Cytokine mRNAs expressed 20 tumor cell lines on mRNAs were expressed in eleven Cell lines;The mRNAs in eight cell lines. Conclusion:The preliminary results suggested that RT-PCR and RNA hybridizationnight be used to test Th1/Th2 cytokine gene expression.