Specific PCR Identification of Hibisci Cortex and Its Adulterants Based on DNA Signature Sequence Tags / 中国实验方剂学杂志

ObjectiveUncommon medicinal herbs are valuable medicinal resources， but their identification is a difficult problem in Chinese medicine due to their particularity and complexity. It is， therefore， urgent to establish a method for the identification of uncommon medicinal herbs. In this study， DNA signature sequence （DSS） tags were used to establish a specific polymerase chain reaction （PCR） identification method for Hibisci Cortex， the origin plant of Hibisci Cortex， and its adulterants. MethodThe candidate DSS tags were obtained from the chloroplast genome sequence analysis， and the DSS tags were verified by DNA sequencing. The specific identification primers for H. syriacus were designed based on the obtained reliable DSS tags. The PCR reaction conditions were optimized， and the tolerance and feasibility were investigated. ResultA DSS tag for identification of H. syriacus was obtained from the comparison of sequencing results of the amplified products with DSS， which revealed the distinguishing characteristics of Hibisci Cortex and its adulterants. A pair of specific primers for H. syriacus was designed according to the DSS tag. After PCR amplification and gel electrophoresis with the primers， a single bright band of about 270 bp was observed from H. syriacus， which did not appear in the four adulterants. ConclusionA DSS tag obtained in this study can be used to identify H. syriacus. The specific primers designed based on this DSS tag can accurately and simply identify the original plant of Hibisci Cortex and its adulterants， which provides a new method and idea for the molecular identification of genuine and counterfeit products of Hibisci Cortex.

Identification of Murrayae Folium et Cacumen by Multiplex Allele-Specific PCR / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction（PCR） method to accurately discriminate the crude materials of Murrayae Folium et Cacumen， Murraya exotica and M. paniculata. MethodBased on the difference in chloroplast genome sequences of M. exotica and M. paniculata， species-specific identification primers P03 and P04 of M. exotica and M. paniculata were designed according to single nucleotide polymorphism （SNP） on the chloroplast genome. A multiplex allele-specific PCR identification method was established for the identification of M. exotica and M. paniculata following the optimization of annealing temperature， number of cycles， and primer concentration ratio. The established PCR method for identification was explored and verified in terms of tolerance and feasibility by investigating the type of Taq polymerases and PCR system model. ResultIn this multiplex allele-specific PCR identification method， about 330 and 230 bp of specific fragments were amplified from DNA templates of M. exotica and M. paniculata， respectively， under the following conditions：cycle number of 31， annealing temperature of 60 ℃， and primer concentration ratio of P03 and P04 of 1∶2. Consistent results were obtained for samples from different sources. ConclusionThe multiplex allele-specific PCR identification method established in this study can accurately identify the origin of Murrayae Folium et Cacumen， which can be used for the simultaneous identification of M. exotica and M. paniculata by the length of fragments in a single identification assay.

Correlation between the Polymorphism of Coagulation-Related Genes and Lower Extremity Deep Venous Thrombosis / 法医学杂志

Objective To investigate the correlation between the polymorphism of 4 coagulation-related genes, rs1799963 （coagulation factor V gene Leiden）, rs6025 （prothrombin gene G20210A）, rs1042579 （thrombomodulin protein gene c.1418C>T） and rs1801131 （methylenetetrahydroflate reductase gene） and lower extremity deep venous thrombosis （LEDVT）. Methods The 4 genotypes mentioned above of 150 LEDVT patients and 153 healthy controls were detected by the kompetitive allele specific polymerase chain reaction （KASP）, then related blood biochemical indicators were collected, binary Logistic regression was established to screen the independent risk factors of LEDVT, and the correlation between polymorphism of 4 coagulation-related genes and LEDVT and its indicators under different genetic modes after adjusting confounding factors were analyzed. Results Five variables, D-dimer, fibrinogen degradation product, homocysteine, sex and age might be the risk factors of LEDVT. These variables were put into 4 genetic inheritance models, and adjusted in binary Logistic regression. The results suggested that the mutations of rs1042579 were correlated with LEDVT under dominant inheritance mode. Conclusion The gene polymorphism of rs1799963, rs6025 and rs1801131 has no significant correlation with the formation of LEDVT. The gene polymorphism of rs1042579 plays a role under dominant inheritance mode, and might be an independent risk factor for formation of LEDVT.

Construction of SNP-STR Multiplex Amplification System with Genetic Markers and Its Forensic Application / 法医学杂志

Objective To select and develop a SNP-STR multiplex amplification system with genetic markers compatible with current STR databases. To understand its genetic polymorphisms in Sichuan Han population and its application value in DNA mixture analysis. Methods Based on the STR genetic markers in commercial kits, SNPs adjacent to these STR markers were selected to be SNP-STR genetic markers. A SNP-STR multiplex amplification system with genetic markers based on allele-specific amplification was constructed using allele-specific amplification primers. The genetic polymorphism of the system in the Sichuan Han population was investigated and the efficiency of systems with different numbers of loci to detect the two individual DNA mixture samples was evaluated. Results An allele-specific multiplex amplification system constituted of 13 SNP-STR genetic markers was selected and constructed. In Sichuan Han population, the heterozygosity of each locus ranged from 0.76 to 0.88, and the combined discrimination power reached 0.999 999 999 999 999 968. In the analysis of the two individual DNA mixture samples： for single-locus amplification, the genotype of the minor components can still be detected when the mixture ratio reaches 1 000∶1; for multiple loci multiplex amplification, the maximum mixture ratio can reach 500∶1. As the number of loci in the system increased, the detection efficiency of the minor components in the DNA mixture decreased. Conclusion SNP-STR genetic markers have a higher polymorphism than STR. The multiplex amplification system made of SNP-STR genetic markers has a better analysis efficiency for mixed samples than traditional STR multiplex amplification system.

Identification of Pheretima by Multiplex Allele-specific PCR / 中国实验方剂学杂志

Objective: In recent years,with the increase in the commodity price of medicinal pheretima,there have emerged increasing adulterates in the medicine market. Besides,the medicinal materials have mostly lost the main identification features, and are difficult to distinguish. Therefore,it is urgent to establish an accurate and stable method for the identification of pheretima. Method: According to the differences of COI gene DNA sequences among Pheretima aspergillum,Pheretima vulgaris,Pheretima guillelmi,Pheretima pectinifera and adulterants,the variation site was found,the specific primers were designed,the reaction conditions were optimized,and the polymerase Chain reaction(PCR) method for identification was explored and verified in terms of tolerance and feasibility in this study. The specific primers were combined to build multiple PCR systems. An effective,accurate,convenient,highly specific and repeatable Multiplex Allele-Specific PCR identification method was established for identifying medicinal pheretima and its common adulterants. Result: Through the established multiplex PCR reaction system, 366,487,487 and 475 bp of fragments were amplified from DNA templates of P. aspergillum,P. vulgaris,P. guillelmi and P.pectinifera respectively. All of the adulterants were negative by the multiplex PCR assay. The PCR amplification of specific alleles method established in this paper can accurately identify pheretima. Conclusion: The Multiplex Allele-Specific PCR identification method established in this paper can accurately identify medicinal pheretima and its adulterants.

Identification of Dendrobium huoshanense, Dendrobium officinale and Dendrobium devonianum by multiplex allele-specific polymerase chain reaction / 药学学报

This study was designed to establish a multiplex allele-specific polymerase chain reaction method for simultaneous identification of Dendrobium huoshanense, D. officinale and D. devonianum, which may resolve identification problems of caulis dendrobii. Internal transcribed spacer sequences and trnL-trnF sequences of the Dendrobium species were aligned by BioEdit software, then specific SNPs of the three species were analyzed for designing allele-specific primers and the multiplex allele-specific PCR reaction system was established. The different origin of Dendrobium huoshanense, D. officinale and D. devonianum was amplified and identified by the sizes of respective band. The results showed that 584 bp, 397 bp and 211 bp bands could be amplified by D. devonianum, Dendrobium officinale and Dendrobium huoshanense respectively, when the annealing temperature was 61 ℃ and the number of cycles was 35. The limit of detection (LOD) of D. devonianum and D. huoshanense were both 1.2 ng, while D. officinale was low than 0.24 ng. The detection limit of adulterates in D. devonianum, D. devonianum and D. huoshanense mixture sample was 1%, 1% and 5% respectively. This result suggests that the method of multiplex allele-specific PCR is useful to identify D. huoshanense, D. officinale and D. devonianum is accurate and specific.

Identification of Panax ginseng, P. notoginseng and P. quinquefolius admixture by multiplex allele-specific polymerase chain reaction / 中国中药杂志

To achieve a molecular method to identify Panax ginseng, P. notoginseng,P. quinquefolius and their admixture. The ITS,18S and matK sequences of Panax genus were analyzed to develop species-specific SNP marker. Three pairs of species-specific primers were designed to establish a multiplex allele-specific polymerase chain reaction (MAS-PCR) and the samples from different region were tested. The results showed that when the annealing temperature was 60 ℃ and the cycle number was 35, approximately 250, 500,1 000 bp specific band were obtained from P. ginseng, P. notoginseng and P. quinquefolius obtain, respectively. This method could also be used to authentificate admixture samples and could detect 0.5% percent of P. notoginseng or P. quinquefolius adulterated in P. ginseng, or 0.5% percent of P. ginseng or P. quinquefolius adulterated in P. notoginseng. The detect limit of P. ginseng in P. quinquefolius was 0.5% and P. notoginseng in P. quinquefolius was 1%. This results showed that the present method could be used as a promise method to identify Panax ginseng, P. notoginseng, P. quinquefolius and their admixture.

A simple, rapid and economic method for detecting multidrug-resistant tuberculosis

OBJECTIVE: To evaluate multiplex allele specific polymerase chain reaction as a rapid molecular tool for detecting multidrug-resistant tuberculosis. METHODS: Based on drug susceptibility testing, 103 isolates were multidrug-resistant tuberculosis and 45 isolates were sensitive to isonicotinylhydrazine and rifampin. Primers were designed to target five mutations hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and multiplex allele specific polymerase chain reaction was performed. Whole-genome sequencing confirmed drug resistance mutations identified by multiplex allele specific polymerase chain reaction. RESULTS: DNA sequencing revealed that 68.9% of multidrug-resistant strains have point mutations at codon 315 of the katG gene, 19.8% within the mabA-inhA promoter, and 98.0% at three hotspots within rpoB. Multiplex allele specific polymerase chain reaction detected each of these five mutations, yielding 82.3% sensitivity and 100% specificity for isoniazid resistance, and 97.9% sensitivity and 100% specificity for rifampin resistance as compared to drug susceptibility testing. CONCLUSIONS: The results show that multiplex allele specific polymerase chain reaction is an inexpensive and practical method for rapid detection of multidrug-resistant tuberculosis in developing countries.

Rapid ABO Genotyping Using Whole Blood without DNA Purification / 대한진단검사의학회지

BACKGROUND: ABO genotyping is commonly used in cases of an ABO discrepancy between cell typing and serum typing, as well as in forensic practice for personal identification and paternity testing. We evaluated ABO genotyping via multiplex allele-specific PCR (ASPCR) amplification using whole blood samples without DNA purification. METHODS: A four-reaction multiplex ASPCR genotyping assay was designed to detect specific nucleotide sequence differences between the six ABO alleles A101, A102, B101, O01, O02, and cis-AB01. The ABO genotypes of 127 randomly chosen samples were determined using the new multiplex ASPCR method. RESULTS: The genotypes of the 127 samples were found to be A101/A102 (n=1), A102/A102 (n=9), A101/O01 (n=3), A102/O01 (n=12), A102/O02 (n=14), B101/B101 (n=5), B101/O01 (n=18), B101/O02 (n=15), O01/O01 (n=14), O02/O02 (n=8), O01/O02 (n=14) and A102/B101 (n=14), from which phenotypes were calculated to be A (n=39), B (n=38), O (n=36) and AB (n=14). The multiplex ASPCR assay results were compared with the serologically determined blood group phenotypes and genotypes determined by DNA sequencing, and there were no discrepancies. CONCLUSIONS: This convenient multiplex ASPCR assay, performed using whole blood samples, provides a supplement to routine serological ABO typing and might also be useful in other genotyping applications.

A/G Gln20Arg (exon 1) and G/A Val156Met (exon 5) polymorphisms of the human orosomucoid 1 gene in Mexico

The human orosomucoid 1 gene (ORM1) codes an alpha-1-acid glycoprotein that has been classified as an acute-phase reactive protein, and a major drug-binding serum component, as well as an immunomodulatory protein with genetic polymorphisms. Evaluation of ORM variation through isoelectric focusing and immunobloting has revealed a world-wide distribution of the ORM1 F and ORM1 S alleles. We evaluated and examined the genetic characteristicsof two Mexican populations that have different anthropological and cultural antecedents, examining two ORM1 genotypes (exon 1 - A/G (Gln20Arg) and exon 5 G/A (Val156Met)) in 145 individuals, using nested polymerase chain reaction, sequencing, and restrited fragment length polymorphism. Mexican Mestizos had higher frequencies of the exon 1 A allele (P = 0.020) and AA genotype(P = 0.018) and lower frequency of the G allele (P = 0.020) when compared to Teenek Amerindians. When we examined exon 5 G/A (Val156Met) polymorphisms, we found significantly higher frequencies of the G allele (P = 0.0007) and the GG genotype (P = 0.0003) in the Mexican Mestizo population. The Teenek population had a significantly higher frequency of the A allele than has been reported for Chinese and African (P < 0.05) populations, and the G/A genotype was more frequently found in this Mexican population than in Chinese, African and European populations (P < 0.05).

A Simplified Approach for Detecting Homologous Deletion of SMN1 Genes in Spinal Muacular Atrophy / 医学研究杂志

Objective To develop a rapid,reliable and convenient approach for diagnosing the homozygous deletion of SMN1 gene.Methods SMN1 gene was amplified specifically with double allele-specific PCR(AS-PCR).Meanwhile,one inrelevant gene was amplified as internal control by PAGE and agarose gel electrophoresis analysis to determine whether the sick children were with homozygous deletion of SMN1 genes.Results The homozygous deletion of exon7 in SMN1 gene was identified by agarose gel electrophoresis or PAGE accurately.Conclusion Compared to PCR-RFLP and DHPLC used in the past,this approach can diagnose homozygous deletion of SMA much more accurate,easier and more convenient without completed following analyses.

The Genotype Frequencies of Platelet-Specific Antigens in Koreans / 대한수혈학회지

BACKGROUND: The platelet-specific antigens which exist specifically on platelets have their antigenic determinant on platelet membrane glycoprotein. The alloantibodies against this antigens are responsible for neonatal alloimmune thrombocytopenia, posttransfusion purpura and platelet refractoriness in multitransfused patients. Discovering of the fact that the polymorphism of platelet-specific antigens is resulted from a single base pair substitution of genomic DNA stimulated studies on genotyping of platelet-specific antigens on various populations. This study was performed to investigate the genotype frequency of platelet-specific antigens in Koreans. METHODS: Using genomic DNA extracted from venous blood of 200 Koreans, genotype of seven platelet-specific antigen systems were determined through allele-specific polymerase chain reaction and restriction fragment length polymorphism. RESULTS: The genotype frequencies of HPA-1 were a+b- 98.0%, a+b+ 1.5%, a-b+ 0.5%; HPA-2, a+b- 85.5%, a+b+ 13.5%, a-b+ 1.0%; HPA-3, a+b- 28.5%, a+b+ 54.0%, a-b+ 17.5%; HPA-4, a+b- 98.0%, a+b+ 2.0%, a-b+ 0.0%; HPA-5, a+b- 95.5%, a+b+ 4.5%; HPA-6W, a+b- 96.0%, a+b+ 4.0%; HPA-7W were a+b- 100.0%, a+b+ 0.0%. CONCLUSION: The gene frequency of HPA-1b in Koreans was lower than that of Caucasian. As a whole, the genotype frequencies of platelet-specific antigens in Koreans were similar to those of the Japanese. However, we found one HPA-1(a-b+) among 200 Koreans, which is very rare in Japanese. This study will serve as a basic data for the study and management of the patients with diseases associated with platelet-specific antigens and antibody reactions in Koreans.