Trionycis Carapax is a commonly used animal medicine in Chinese medicine. It's difficult to identify Trionycis Carapax and its adulterants because of the loss of morphological characteristics after processing. To establish an efficient and stable method to identification Trionycis Carapax, this study combines SDS method with column purification to extract genomic DNA, uses universal primers for polymerase chain reaction (PCR) amplification and sequencing, and designs the specific primers based on the differences in the sequences of Pelodiscus sinensis and their adulterants. When the annealing temperature was 62 °C and the number of cycles was 35, the designed primer Biejia-272.F/R was used for PCR amplification and got optimum results. The crude drug and preparation of P. sinensis were all amplified to obtain a specific band of approximately 300 bp, while the adulterants showed no such a band. This method can be used as a rapid and accurate method to identify the authenticate of P. sinensis.
Animals ; DNA Primers ; Polymerase Chain Reaction

Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction (mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA, five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei (H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155, 222, 292, 352, 458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.
Animals ; DNA Primers ; DNA, Mitochondrial ; Multiplex Polymerase Chain Reaction ; Smegmamorpha

Obvious epigenetic differentiation occurred on Lycium barbarum in different cultivation areas in China. To investigate the difference and change rule of DNA methylation level and pattern of L. barbarum from different cultivation areas in China, the present study employed fluorescence-assisted methylation-sensitive amplified polymorphism(MSAP) to analyze the methylation level and polymorphism of 53 genomic DNA samples from Yinchuan Plain in Ningxia, Bayannur city in Inner Mongolia, Jingyuan county and Yumen city in Gansu, Delingha city in Qinghai, and Jinghe county in Xinjiang. The MSAP technical system suitable for the methylation analysis of L. barbarum genomic DNA was established and ten pairs of selective primers were selected. Among amplified 5'-CCGG-3' methylated sites, there were 35.85% full-methylated sites and 39.88% hemi-methylated sites, showing a high degree of epigenetic differentiation. Stoichiometric analysis showed that the ecological environment was the main factor affecting the epigenetic characteristics of L. barbarum, followed by cultivated varieties. Precipitation, air temperature, and soil pH were the main ecological factors affecting DNA methylation in different areas. This study provided a theoretical basis for the analysis of the epigenetic mechanism of L. barbarum to adapt to the diffe-rent ecological environments and research ideas for the introduction, cultivation, and germplasm traceability of L. barbarum.
China ; DNA Methylation ; DNA Primers ; Epigenesis, Genetic ; Lycium/genetics*

Andrographis Herba, the aerial part of Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae), has a wide geographic distribution and has been used for the treatment of fever, cold, inflammation, and other infectious diseases. In markets, sellers and buyers commonly inadvertently confuse with related species. In addition, most Chinese medicinal herbs are subjected to traditional processing procedures, such as steaming and boiling, before they are sold at dispensaries; therefore, it is very difficult to identify Andrographis Herba when it is processed into Chinese medicines. The identification of species and processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA barcoding has received considerable attention as a new potential means to identify species and processed medicinal materials. In this study, 17 standard reference materials of A. paniculata, 2 standard decoctions, 27 commercial products and two adulterants were collected. Based on the ITS2 sequence, it could successfully identify A. paniculata and adulterants. Moreover, a nucleotide signature consisting of 71 bp was designed, this sequence is highly conserved and specific within A. paniculata while divergent among other species. Then, we used these new primers to amplify the nucleotide signature region from processed materials. In conclusion, the DNA barcoding method developed in the present study for authenticating A. paniculata is rapid and cost-effective. It can be used in the future to guarantee the quality of Andrographis Herba of each regulatory link for clinical use.
Andrographis ; Andrographis paniculata ; DNA Primers ; Drugs, Chinese Herbal

Bacillus anthracis is a soil pathogen capable of causing anthrax in animals and humans. To establish a method for specifically detecting B. anthracis, we used nested polymerase chain reaction. Outer and inner sets of oligonucleotide primers were designed from the protective antigen (pag) gene and from the cya gene of the plasmid pXO1. Ainplification of 482 bp or 208 bp DNA fragment obtained from a nested PCR method provided the basis for rapid and reliable assay for the detection and identification of B. anthracis.
Animals ; Anthrax ; Bacillus anthracis* ; Bacillus* ; DNA ; DNA Primers ; Humans ; Plasmids ; Polymerase Chain Reaction* ; Soil

BACKGROUND: Vibrio parahaemolyticus is one of the most important food-borne pathogens in Korea. Although the mechanism of its pathologic effect is still not clearly understood, epidemiological studies have suggested a very strong association of thermostable direct hemolysin (TDH) and thermostable direct hemolysin-related hemolysin (TRH) with the disease. We detected the tdh gene and the trh gene of V. parahaemolyticus isolates by polymerase chain reaction (PCR). METHODS: V. parahaemolyticus strains were isolated from clinical specimens of patients with diarrhea in different geographic areas of Seoul (16 cases), Inchon (27 cases) and Kwang Joo (2 cases) in Korea between 1998 and 2000. The colonies selected were identified by using the API 20E identification strip (API system, Analytab Products, Montalieu-Vercieu, France). PCR protocols were established for specific detection of the tdh and trh genes. Oligonucleotide primers were designed based on the reported nucleotide sequence of the tdh2 gene and the trh1 gene, respectively. RESULTS: The protocols established for the tdh and trh genes could detect 400 fg (100 colony-forming units) of cellular DNA carrying the respective gene. All strains of V. parahaemolyticus isolates in this study contained the tdh gene but not the trh gene. CONCLUSIONS: The detection of the tdh gene and trh gene of V. parahaemolyticus using the PCR is an easy and rapid method.
Base Sequence ; Diarrhea ; DNA ; DNA Primers ; Humans ; Incheon ; Korea ; Polymerase Chain Reaction* ; Seoul ; Vibrio parahaemolyticus* ; Vibrio*

To investigate the genetic diversity among wild Dipsacus asperoides in China, 66 germplasmic resources of D. asperoides were analyzed by Start Codon Targeted Polymorphism (SCoT) molecular markers. Genetic distance was calculated by TREECONW software and the systematic diagram of genetic relationship was clustered by UPGMA method. The results showed that the totals of 181 bands were detected using 20 primers , among which 109 were polymorphic bands. The average percentage of polymorphic bands was 60.13%. Genetic distance changed from 0.030 6 to 0.181 4. The clustering results showed that there was no significant correlation between the classification of the wild D. asperoides and their geographical origin. The relatively high genetic diversity of D. asperoides provides the basis for breeding new varieties.
China ; DNA Primers ; genetics ; DNA, Plant ; genetics ; Dipsacaceae ; chemistry ; classification ; genetics ; Genetic Variation ; Phylogeny ; Polymorphism, Genetic

OBJECTIVETo study the identification method of Syngnathus and its adulterants.

METHODRandom amplified polymorphic DNA (RAPD) was used to construct a dendrogram by UPGMA method based on Nei & Li's coefficient and a genetic affinity pattern for Syngnathus acus, Solenognathus hardwickii, Syngnathoides biaculeatus, Trachyrhamphus serratus, Halicampus koilomatodon, Microphis boaja.

RESULTFour primers, LJ04, LJ09, LJ16 and LJ19, from 18 random primers were used in the dendrogram which can differentiate Syngnathus in genus level and showed a great consistence with the appearance identification. The genetic affinity pattern based on primers LJ09 and LJ19 could be used to identify Syngnathus from its adulterants.

CONCLUSIONRAPD is suitable to identify Syngnathus and its adulterants.

Animals ; China ; DNA Primers ; genetics ; Phylogeny ; Polymorphism, Genetic ; Random Amplified Polymorphic DNA Technique ; Smegmamorpha ; classification ; genetics

To evaluate the roles of 8 short tandem repeats (STR) loci as STR panel in quantitative analysis of chimerism following transplantation, the primers were synthesized and marked with different dyes for D3S3045, D4S2366, D4S2639, D5S818, D13S317, D18S1002, D20S481 and D22S689. The blood samples of 15 cases received allogeneic stem cell transplantation were collected before and after transplantation, then DNA was extracted and amplified with these primers, and was further analysed under ABI Genetic Analyser 3100 to select suitable informative STR locus. Donor/recipient dilution series were prepared to get standard curves in selected loci, the DNAs extracted at different days after transplantation were used to quantitatively analyze the chimerism in patients according to the values of peak area or peak height of fluorescent signals. The standard curves can be used to calculate the chimerism by plotting the respective R/D quotient value against the percentage of recipient DNA. The results indicated that the calculated chimerism was in concordance with the donor/recipient dilution. The STR panel succeeded in identifying at least one informative marker and quantitative monitoring the chimerism after HSCT in 15 donor-recipient pairs and a relapsed case was diagnosed. It is concluded that the STR panel and its detection method can accurately and quantitatively monitor the chimerism after allogeneic HSCT, which is more economical and flexible than using commercial kits.
DNA ; genetics ; DNA Primers ; Hematopoietic Stem Cell Transplantation ; Humans ; Microsatellite Repeats ; Transplantation Chimera ; genetics

The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.
Archaea/genetics* ; Bacteria/genetics* ; DNA Primers ; DNA, Bacterial ; Humans ; Microbiota/genetics* ; Phylogeny ; RNA, Ribosomal, 16S/genetics*