Identification of Cervus nippon, C.elaphus and their hybridize samples based on COI and SRY gene / 中国中药杂志

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.

Rapid PCR identification of Houttuynia cordata and Gymnotheca chinensis / 中草药

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.

Analysis of the usage of TCRβ V/J subfamily and CDR3 repertoire in patients with peripheral T-cell lymphoma unspecified / 白血病·淋巴瘤

Objective To analyze the restricted usage of TCRβ V/J subfamily and CDR3 repertoire in patients with peripheral T-cell lymphoma unspecified (U-PTL).Methods The total RNA was respectively extracted from lymph node of U-PTL and reverse transcriptase,then multi-PCR was used to amplify the complete DNA sequence(CDS) of TCR β-chain.The recombinant plasmids were sequenced and sequence was analyzed by using online TCR resources.Results There were 9 TCR β chain CDS obtained from four patients.TCRβ-chain presented specific repertoire skewing in patients with U-PTL.There were restricted usage of BV2,BV4S2,BV14,BV29S1 of BV subfamily and BJ1S4,BJ2S3,BJ2S5,BJ2S7 of BJ subfanily.The clonal proliferation T cells had different CDR3 amino acid sequences.Conclusions There are restricted usage of TCR β V/J subfamily in patients with U-PTL.The sequences of CDR3 in different TCR clone proliferation are mostly different.

Establishment and application of multi-PCR-SSCP assay for rapid detection of isoniazid- and rifampin-resistance Mycobacterium tuberculosis isolates / 中华微生物学和免疫学杂志

Objective To establish multi-PCR-single strand conformational polymorphism analysis (mPCR-SSCP) for rapid detection of isoniazid (INH) and rifampin (RIF) resistance associated katG,inhA and rpoB genes of Mycobacterium tuberculosis isolates.Methods The INH-and RFP-resistance of 134 isolates was determined by using drug susceptibility test.The primers were designed for detecting INH and RFP resistance-associated katG,inhA and rpoB gene in the isolates by mPCR-SSCP.PCR-DS technique was applied to detect the mutations in katG,inhA and rpoB genes.All the results from different assays were subsequently analyzed as well as compared.Results All of the 134 tested isolates had katG,inhA and rpoB genes.Of the 134 isolates,42 (31.3％) and 45 (33.6％) strains were INH-and RFP-resistant,respectively.The results of mPCR-SSCP and PCR-DS showed that all the 92 INH-susceptible isolates had no mutation in katG and inhA genes with 100％ specificity.In the 89 RFP-susceptible isolates,2 and 1 had mutation in rpoB genes confirmed by mPCR-SSCP and PCR-DS with 97.8％ or 98.9％ specificity,respectively.Among the 42 INH-resistance isolates,33 and 36 strains had the mutations in katG and/or inhA genes due to the results of mPCR-SSCP and PCR-DS with 78.6％ or 85.7％ sensitivity,respectively.The results of mPCR-SSCP and PCR-DS also demonstrated that in the 45 RFP-resistance isolates,41 and 43 strains had the mutations in rpoB gene with 91.1％ or 95.6％ sensitivity,respectively.Conclusion The mPCR-SSCP established in this study can be used to rapidly detect INH and RFP-resistance associated mutations in katG,inhA and rpoB genes of M.tuberculosis with convenience,specificity and sensitivity,which shows a good prospect for application in clinic.

Detection and Significance of Class Ⅱ Integrons in Clinically Isolated Strains of Stenotrophomonas maltophilia / 中华医院感染学杂志

OBJECTIVE To investigate the distribution and characterization of the classes Ⅰ,Ⅱ and Ⅲ integrons on Stenotrophomonas maltophilia and clarify their influence on the bacterial drug-resistance.METHODS A multi-PCR assay using specific primers of int1,int2 and int3 was constructed to screen classes Ⅰ,Ⅱ and Ⅲ integrons.RESULTS Class Ⅰ integron was detected in 13.4% of clinical isolates,3 isolates harbored among class Ⅱ integrons. There was not been reported in abroad.CONCLUSIONS Classes Ⅰ and Ⅱ integrons could play an important role in causing the antibiotic multidrug resistance.

Development of autosomal SNP fluorescent-multiplex by the method of fragment length discrepant allele specific PCR / 中国法医学杂志

Objective To develop autosomal SNP fluorescent-multiplex PCR system,and investigate the allele frequencies of these 13 SNP loci in Southern Liaoning Han population.Methods After selected 13 autosomal SNP loci,SNP fluorescent-multiplex system was developed based on the principle of fragment length discrepant allele specific PCR,and the allele frequencies of these SNP loci in Southern Liaoning Han population were investigated using the established system.Results For the same SNP locus,the homozygote showed a single product peak,and the heterozygote showed two product peaks with different length.For the different SNP loci,the lengths of their PCR products were different.Therefore,we could genotype the 13 SNP loci simultaneously according to the length of products and the amount of product peaks,and the results were in accordance with that of direct sequencing.In the meanwhile,the allelic frequencies of these 13 SNP loci in Southern Liaoning Han population were obtained.Conclusion The SNP fluorescent-multiplex system based on the fragment length discrepant allele specific PCR strategy is simple and economical,and is of a high application value in forensic medicine.

Analyses for the ?/? T Cell Receptor Gene Rearrangement and CDR3 Repertoire in Active Pulmonary Tuberculosis Patients / 中国免疫学杂志

Objective:To establish a method of multi-PCR to amplify the complete DNA sequence (CDS) of TCR ? and ? chain of the antigen-specific T lymphocytes in local pathologic specimen of active pulmonary tuberculosis patients, and to analyze ?/? T cell receptor gene rearrangement and CDR3 repertoire.Methods:The lymphocytes in bronchoalveolar lavage (BAL) of active pulmonary tuberculosis patients were separated. Following total RNA extraction, cDNA synthesis, Multi-PCR, recombinant clones construction, and sequencing, the CDS of TCR ? and ? chains from these lymphocytes were analyzed by using software of DNAstar and internet TCR resources.Results:24 of ? chain CDS and 13 of ? chain CDS from 3 samples of BAL were obtained. As for TCR ? chain, AV1S2 (54%), AV12S3 (41%), and AV12S2(5%) appeared frequently. BV2(38%), BV29S1(46%), BV14(3%), and BV4S2(3%) in TCR ? chain appeared more often. There were CDR3 diversities between samples and even in the same sample by amino acid sequence analysis, but there were a few identical or similar amino acid sequences. There was the same amino acid sequence of SVGTGTLHQETQY in CDR3 region of ? chain of BAL sample No.1 and No.2; The sequence of AVRDWAGNMLT appeared in two ? chains of BAL sample No.2 and No.3; Moreover, the sequence of AV…DNN…RLM appeared in ? chains of BAL sample No.2 and No.3.Conclusion:A method of Multi-PCR is used to amplify TCR ? and ? chain CDS of tuberculosis patients. There are characteristic T cell clones to proliferate,with TCR ? and ? chain repertiore skewing in local infective focus. The sequences of CDR3 in different TCR clones are mostly different but there are a few identical or similar sequences in the same patient or even between different patients. The identical amino acid sequences of CDR3 are possibly specific for recognizing MTB polypeptide.