Rapid Screening of Gastrodia elata with High Purity by PCR-RFLP Identification / 中国实验方剂学杂志

ObjectiveTo establish a rapid screening method for germplasm materials of Gastrodia elata with high purity， and lay a foundation for pure line breeding and cross breeding. MethodBased on the whole genome sequencing and population resequencing of G. elata， 20 restriction fragment length polymorphism (RFLP) markers were developed by single nucleotide polymorphism (SNP) sites. The polymerase chain reaction (PCR)-RFLP method was used to carry out restriction endonuclease experiments on 20 RFLP markers of 15 G. elata germplasms. According to the number of enzymatic bands at 20 RFLP marker sites, the purity of 15 germplasms was calculated and evaluated. On this basis, genome resequencing technology was used to verify the assessment results. ResultTen germplasm materials with purity greater than 95% were screened out by PCR-RFLP method， 3 of which had 95% purity and 7 had 100% purity. Nine germplasm materials with purity greater than 95% were screened out by genome resequencing methods, and 8 of them were consistent with the results of PCR-RFLP. ConclusionThe PCR-RFLP method established in this study for screening G. elata germplasms with high purity precision of RFLP markers has 80% precision and 89% accuracy. The method is simple， efficient， and significantly less expensive than genome resequencing method, which provides technical support for pure line breeding of G. elata and references for breeding of other Chinese medicinal materials.

A Case of Mycobacterium Marinum Tenosynovitis Diagnosed by the PCR-restriction Fragment Length Polymorphism of the rpoB Gene / 대한내과학회지

Mycobacterium marinum is an uncommon cause of skin and soft-tissue infection. The diagnosis of M. marinum infection is often delayed when only a conventional tissue culture method is used. PCR-restriction fragment length polymorphism (RFLP) analysis using the novel region of the rpoB gene is now available for the rapid identification of Mycobacteria. We report a case of hand infection caused by M. marinum that was identified by PCR-RFLP analysis. The PCR-RFLP assay is a specific and rapid method for the identification of Mycobacteria that facilitates the early diagnosis of non-tuberculous Mycobacterium infection.

A Case of Mycobacterium Marinum Tenosynovitis Diagnosed by the PCR-restriction Fragment Length Polymorphism of the rpoB Gene / 대한내과학회지

Mycobacterium marinum is an uncommon cause of skin and soft-tissue infection. The diagnosis of M. marinum infection is often delayed when only a conventional tissue culture method is used. PCR-restriction fragment length polymorphism (RFLP) analysis using the novel region of the rpoB gene is now available for the rapid identification of Mycobacteria. We report a case of hand infection caused by M. marinum that was identified by PCR-RFLP analysis. The PCR-RFLP assay is a specific and rapid method for the identification of Mycobacteria that facilitates the early diagnosis of non-tuberculous Mycobacterium infection.

Relationship Between gyrA Gene Mutations in Clinical Isolates of Pseudomonas Aeruginosa and Quinolone Resistance / 中国药房

OBJECTIVE:To observe the relationship between gyrA gene mutations of the clinical isolates of Pseudomonas aeruginosa and quinolone resistance and to evaluate the feasibility of analyzing gyrA gene mutations using PCR-RFLP-SSCP.METHODS:With gyrA gene order of the clinical isolates of Pseudomonas aeruginosa taken as target sequence,gyrA gene mutations in strain ATCC 27853 and 16 clinical isolates of Pseudomonas aeruginosa were analyzed contrastively using PCR,PCR-RFLP,PCR-SSCP,and DNA sequencing.RESULTS:Of the total 8 ciprofloxacin resistant Pseudomonas aeruginosa,6 strains showed single point ACC→ ATC mutation in the gyrA gene at codon 83,leading to amino acid substitution of Thr83→Ile.SacⅡ digestion fragment of the PCR amplified products in gyrA gene was in line with the sequencing results.SSCP showed that the banding patterns of all strains were different from that of strain ATCC 27853 except 2 strains.CONCLUSION:The molecular mechanism of the quinolone resistant Pseudomonas aeruginosa isolated from clinics was manifested as mutations in the gyrA gene at codon 83.The results showed that PCR-RFLP-SSCP is a rapid and accurate method for the detection of basyl variation in gyrA in quinolone resistant Pseudomonas aeruginosa.

Factor V Leiden mutation in Korean women with pregnancy-induced hypertension / 대한산부인과학회지

OBJECTIVE: The purposes of this study was to evaluate the frequency of Leiden mutation (missense mutation in the factor V gene at exon 10, 1691 CGA to CAA) in Korean women with well characterized pregnancy-induced hypertension (PIH) compared with normotensive gravid women. METHODS: Genomic DNA from 121 PIH cases and 98 normotensive pregnant control cases were used for polymerase chain reaction (PCR). To genotype Leiden mutation (missense mutation in the factor V gene, exon 10 (1691 G to A)), primers (5'-TGC CCA GTG CTT AAC AAG ACC A-3', 5'-TGT TAT CAC ACT GGT GCT AA-3') were employed to make 267 base pair (bp) PCR product. There was an initial denaturation at 94 degrees C 5 min, followed by 30 cycles of one minute at 94 degrees C, one minute at 55 degrees C, and one minute at 72 degrees C. A 267 bp PCR product was further digested with Mnl I for 2 hour at 37 degrees C and analysed through 12% polyacrylamide gel electrophoresis to determine genotype. Allele 1691G yielded 37 bp, 67 bp, 163 bp fragment and allele 1691A yielded 67 bp, 200 bp fragment. RESULTS: We examined the genotypes of factor V of 121 Korean women with pregnant induced hypertension and 98 normal pregnant women. None of the 219 Korean women carried the factor V Leiden mutation. CONCLUSION: The factor V Leiden mutation is absent and not a common cause of PIH in Korean women.

Promoter -202 A/C Polymorphism of Insulin-like Growth Factor Binding Protein-3 Gene and Non-small Cell Lung Cancer Risk / 결핵및호흡기질환

BACKGROUND: IGFBP-3 inhibits the mitogenic and anti-apoptotic activity of IGF by blocking the binding of IGF to its receptor. However, under certain circumstances, IGFBP- 3 can enhance the activity of IGF by protecting IGF from its degradation. More than half of the inter- individual variations in IGFBP-3 levels are known to be genetically determined by the polymorphism at -202 locus of IGFBP-3 gene. METHOD: We attempted to ascertain whether A-202C poly?morphic variation of IGFBP-3 gene constitutes a risk factor for non-small cell lung cancer (NSCLC), using PCR-restriction fragment length polymorphism (RFLP). Our study included 104 NSCLC patients and 104 age-, gender-, and smoking status-matched control subjects. RESULT: In the 104 NSCLC subjects, the genotypic freque?ncies at the -202 site were as follows: AA = 67 (64.4%), AC = 35 (33.7%), and CC = 2 (1.9%). We did detect significant differences in the genotypic distribution between the NSCLC and the control subjects (pAC>CC). Using CC genotype as a reference, the odds ratio (OR) for the subjects with AC genotype was 2.60 (95% CI: 0.89 - 8.60), and the OR associated with AA genotype was 5.89 (95% CI: 1.92 - 21.16). CONCLUSION: These results indicate that the dysregulation of IGF axis should now be considered as another important risk factor for NSCLC, and a potential target for novel antineoplastic therapies and/or preventative strategies in high-risk groups.

Molecular Genetic Alteration and Loss of Genes on the Short Arm of the Chromosome 11 in Bladder Tumor / 대한비뇨기과학회지

Although bladder transitional cell carcinoma (TCC) is common, the underlying molecular events remain ill-defined. So we attempted to define the role of tumor suppressor genes in the pathogenesis of bladder tumor through a molecular genetic study. For 15 bladder TCC (6 gradeII, 1 gradeIII, and 8 grade IV), we performed the restriction fragment length polymorphism (RFLP) analysis for 6 loci of suspected or established tumor suppressor regions (3p21, 3p24-25, llp15, 13q14, and 17p13). Our data confirm that allelic losses are highly common in bladder tumors. We found that alleles from each of the four chromosomal arms tested were lost in most of the tumors. Reduction of allele occured at 3p21 (13%), 3p24~25 (50%), and 13q14 (38%). However, the greatest frequency of allelic loss was seen for 17p 13 (100% of informative cases) and llp15.5 (87% informative cases). Severe allelic losses of chromosome 17p and pADJ762 on lip were seen only in grade IV, not in grade II. Amplification of 3p21 was seen six out of eight. Amplification of 3p21 has not been previously observed on the other study. Addition to this, we observe the loss of H-ras allele on 11p in one case which was associated with duplication of the retained allele as was demonstrated in Wilms'tumors. The results of out study suggest that deletions of pADJ762 on chromosome 11p and 17p13 occur in high grade bladder tumor and may contribute to the progression of this disease. But, there was no apparent correlation between tumor grade and the loss of 3p or 13q14 alleles although they had some deletions. The role of these genetic alterations in the prognosis of bladder transitional cell carcinoma will require additional follow-up and further studies.

Type 2 Procollagen Gene Mutation in Osteoarthritis / 대한류마티스학회지

OBJECTIVES: Human osteoarthritis is a heterogeneous and multifactorial disease characterized by the progressive deterioration of the cartilage of diarthrodial joints. In some instances, there is an identifiable cause, such as trauma or congenital malformation, but, mostly the etiology remains unknown. Since familial aggregation is seen, genetic factors may be important, particularly in osteoarthritis of the hand. METHODS: Blood samples from patients and controls were obtained for DNA analysis. Exon 31 of type II procollagen gene was amplified by polymerase chain reaction, and screening for the mutation was undertaken using a restriction enzyme digestion (Dsa I). RESULTS: The patients phenotype represented typical, but earlyonset and family history, osteoarthritis. No mutation in exon 31 of type II procollagen gene could be identified. CONCLUSION: Screening of the 31 exon did not, however, reveal any mutation. It needs further evaluation in other sites of type II procollagen genes.