Identification of Armeniacae Semen Amarum Adulterated in Persicae Semen by Allele-specific Polymerase Chain Reaction / 中国实验方剂学杂志

Objective:Due to the limitation of traditional identification methods of Chinese medicinal materials， the study established a rapid method to identify Persicae Semen mixed with Armeniacae Semen Amarum by allele-specific polymerase chain reaction （PCR）. Method:By comparing the ribosomal DNA internal transcribed spacer （ITS） gene sequences of Persicae Semen and Armeniacae Semen Amarum， single nucleotide polymorphism （SNP） sites were searched and specific primers were designed. Different Persicae Semen and Armeniacae Semen Amarum samples were amplified by PCR， the effects of annealing temperature， primer concentration and cycle number on the PCR reaction system were optimized， and the specificity and detection limit of this method were investigated. In addition， the established PCR method was used to detect the samples of Persicae Semen mixed with different proportion of Armeniacae Semen Amarum from different sources and producing areas. Result:A specific PCR method for identifying Persicae Semen mixed with Armeniacae Semen Amarum was established. When the annealing temperature was 63 ℃ and the number of primer cycles was 30， only Armeniacae Semen Amarum could be amplified with 432 bp specific band， while Persicae Semen samples did not have this band. The minimum detection limit of this method for Armeniacae Semen Amarum was 0.2 ng， and the detection limit for Armeniacae Semen Amarum adulterated in Persicae Semen was 1%. Conclusion:The established allele-specific PCR method can accurately detect whether there is Armeniacae Semen Amarum in Persicae Semen， which can provide experimental basis for the quality control of Persicae Semen and guarantee the safety of its clinical use.

Rapid detection of CYP2C9, CYP2C19, CYP4F2, VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology / 中华检验医学杂志

Objective To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P4502C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology. Methods Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results. Results The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4％and 10.9％, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results. Conclusion A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.

Rapid detection of CYP2C9, CYP2C19,CYP4F2,VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology / 中华检验医学杂志

Objective@#To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology.@*Methods@#Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results.@*Results@#The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results.@*Conclusion@#A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.

Molecular genotyping of clinically important blood group antigens in patients with thalassaemia

Background & objectives: In multitransfused thalassaemic patients, haemagglutination fails to phenotype the patient's blood group antigens due to the presence of donor-derived erythrocytes. DNA-based methods can overcome the limitations of haemagglutination and can be used to determine the correct antigen profile of these patients. This will facilitate the procurement of antigen-matched blood for transfusion to multitransfused patients. Thus, the aim of this study was to compare the serological phenotyping of common and clinically important antigens of Rh, Duffy, Kell, Kidd and MNS blood group systems with molecular genotyping amongst multitransfused thalassaemic patients. Methods: Blood samples from 200 patients with thalassaemia and 100 'O' group regular blood donors were tested using standard serological techniques and polymerase chain reaction-based methods for common antigens/alleles (C, c, D, E, e, Fya, Fyb, Jka, Jkb, K, k, M, N, S, s). Results: Genotyping and phenotyping results were discordant in 77 per cent of thalassaemic patients for five pairs of antithetical antigens of Rh, Duffy, Kell and Kidd blood group systems. In the MNS blood group system, 59.1 per cent of patients showed discrepancy. The rate of alloimmunization among thalassaemics was 7.5 per cent. Interpretation & conclusions: Molecular genotyping enabled the determination of the actual antigen profile in multitransfused thalassaemia patients. This would help reduce the problem of alloimmunization in such patients and would also aid in the better management of transfusion therapy.

A highly sensitive method for the detection of Chrysanthemum virus B

Background: Chrysanthemum plants are subject to serious viral diseases. The viruses cause severe losses of the quantity and quality of chrysanthemum. The most problematic pathogen of chrysanthemum is typically considered Chrysanthemum virus B (CVB). Thus, a method for the simultaneous detection of CVB is needed. Results: We used gene-specific primers, which were derived from the coat protein gene region of the virus, for reverse transcription to obtain cDNA. Nested amplification polymerase chain reaction (PCR) was employed to detect the viral gene. This method was sensitive enough to detect the virus at up to 10-9 dilution of the cDNA. Conclusion: A highly specific and sensitive nested PCR-based assay has been described for detecting CVB. This new method is highly specific and sensitive for the detection of CVB, which is known to infect chrysanthemum plants in the fields. Further, this protocol has an advantage over traditional methods as it is more cost-effective. This assay is ideal for an early stage diagnosis of the disease.

Detection of First-Line Anti-Tuberculosis Drug Resistance Mutations by Allele-Specific Primer Extension on a Microsphere-Based Platform

BACKGROUND: Resistance of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs is almost exclusively due to spontaneous chromosomal mutations in target genes. Rapid detection of drug resistance to both first- and second-line anti-TB drugs has become a key component of TB control programs. Technologies that allow rapid, cost-effective, and high-throughput detection of specific nucleic acid sequences are needed. This study was to develop a high-throughput assay based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres to detect anti-TB drug resistance mutations. METHODS: DNA samples from 357 M. tuberculosis clinical isolates and H37Rv were amplified by multiplex PCR using four primer sets, followed by multiplex ASPE using 23 TAG-ASPE primers. The products were sorted on the TAG-ASPE array and detected by using the Luminex xMAP system. Genotypes were also determined by sequencing. RESULTS: Genetic drug susceptibility typing by the TAG-ASPE method was 100% concordant with those obtained by sequencing. Compared with phenotypic drug susceptibility testing (DST) as a reference method, the sensitivity and specificity of the TAG-ASPE method were 83% (95% confidence interval [CI], 79-88%) and 97% (95% CI, 90-100%) for isoniazid. For rifampin testing, the sensitivity and specificity were 90% (95% CI, 86-93%) and 100% (95% CI, 99-100%). Also, the sensitivity and specificity were 58% (95% CI, 51-65%) and 86% (95% CI, 79-93%) for ethambutol. CONCLUSIONS: This study demonstrated the TAG-ASPE method is suitable for highly reproducible, cost-effective, and high-throughput clinical genotyping applications.

Identification of Velvet Antler by mitochondrial DNA fingerprint / 中国药学杂志

OBJECTIVE: To compare specific primer method and r andom amplified polymorphic DNA (RAPD) method for identification of Velvet Antler, thus to choose a simpler method. METHODS: DNA samples were extracted from sika deer antler, wilddeer antler, reideer pilose antler and commercially available Velvet Antler, and then purified and amplified with specific primer amplification and RAPD amplification. RESULTS: The sika deer antler, wild deer antler, reideer pilose antler and some of the commercially available Velvet Antler all showed 313bp segment in the agarose gel with different brightness while the other commercial products did not show this segment. RAPD not only effectively showed positive and negative DNA amplification results, but also showed the difference in PCR products of sika deer antlerwild deer antler with different strip numbers and strip brightness. CONCLUSION: RAPD method is more accurate and rapid and it has wide application prospect.

Association of killer cell immunoglobulin-like receptor genes 2DS4 and its variant KIR1D with syphilis / 中华微生物学和免疫学杂志

Objective To investigate the killer cell immunoglobulin-like receptor (KIR) genes, KIR2DS4 and its variant KIR1D for an association with syphilis in the comparison between syphilis patients and unrelated healthy subjects. Methods One hundred and ninety syphilis patients and 192 unrelated healthy subjects were performed to determine the KIR genotypes by PCR-SSP method. The gene frequencies of KIR2DS4 and KIR1D were analyzed for an association with syphilis in the patients and healthy people who belonged to KIR gene haplotype A. Results Of 192 healthy individuals, 187 were identified with a KIR2DS4 gene. And 91 individuals were classified as homozygous haplotype A with the percent of 48.7% (91/187) in 187 KIR2DS4 positive individuals. Of 190 syphilis patients, 181 were identified with a KIR2DS4 gene. And 89 individuals were classified as homozygous haplotype A with the percent of 49.2% (89/181) in 181 KIR2DS4 positive individuals. The frequency of KIR1D/KIR1D in syphilis patients classified as haplotype A was 16.9%, and was significantly higher than that in the control group (6.6%, P=0.032). However, there was no significant difference for the frequencies of KIR2DS4/KIR2DS4 and KIR2DS4/KIR1D between the two groups (P>0.05). Conclusion KIR1D/KIR1D might be associated with syphilis in the comparison between syphilis patients and unrelated healthy controls who were classified as homozygous haplotype A.

Association between atopy for Platanus Acerifolia pollen and HLA-DRB1 alleles / 临床检验杂志

Objective To develop a PCR-SSP method for detection of HLA-DRB1 alleles in the patients who were hypersensitive to Platanus Acerifolia pollen allergen,and to probe into the association between the atopic subjects to Platanus Acerifolia pollen allergen and HLA-DRB1 alleles.Methods DNA in whole blood was extracted by phenol-chloroform method.Eight pairs of specific primers for alleles were synthesized,and HLA-DRB1*0401,*0402,*0403,*0404,*0405,*0406,*0407,*0408 alleles in 20 atopic patients and 36 healthy individuals of Jiangsu Province with Han nationality were detected by PCR-SSP(polymerase chain reaction-sequence specific primer).Results By optimizing the experimental conditions PCR-SSP methods for detection of the 8 alleles were established and the distributing data of above-mentioned HLA DRB1 were obtained.The frequency of HLA DRB1*0405 and *0406 in the patients group was higher than that of in healthy controls group,while the frequency of HLA DRB1*0402 in the patients group was lower than that in controls.No significant deference for the other 5 alleles was found between the 2 groups.Conclusion HLA-DRB1*0406和*0405 seems to be the likely suspected candidate alleles responsible for susceptibility to Platanus Acerifolia pollen allergen in the atopic patients,while DRB1*0402 might be contribute to the related resistance to the allergen.

Molecular Identification of Arbuscular Mycorrhizal Fungal Spores Collected in Korea

Arbuscular mycorrhizas (AM) have mutualistic symbiosis with plants and thus efforts have been placed on application of these symbiotic relationships to agricultural and environmental fields. In this study, AM fungi were collected from 25 sites growing with 16 host plant species in Korea and cultured with Sorghum bicolor in greenhouse condition. AM fungal spores were extracted and identified using both morphological and molecular methods. Using morphological characters, total 15 morpho-speices were identified. DNA was extracted from single spore of AM fungi and a partial region on 18S rDNA was amplified using nested PCR with AM fungal specific primers AML1/AML2. A total of 36 18S rDNA sequences were analyzed for phylogenetic analysis and 15 groups of AM fungi were identified using both morphological and molecular data of spores. Among the species, 4 species, Archaeospora leptoticha, Scutellospora castanea, S. cerradensis, S. weresubiae were described for the first time in Korea and two species in Glomus and a species in Gigaspora were not identified. Morphological and molecular identification of AM fungal spores in this study would help identify AM fungal community colonizing roots.

Detection of Helicobactor pylori by polymerase chain reaction: A comparison in sample preparation.

Gastric biopsy samples obtained from 14 patients with upper abdominal pain, clinically diagnosed as acid peptic disease, were analysed for the presence of Helicobacter pylori (H. pylori) by Polymerase Chain Reaction (PCR) using partially (template A) and completely purified DNA (template B). Antigen specific primer was used to analyse the sample by PCR method. The presence of H. pylori in the samples was confirmed by running a positive control. The presence of H. pylori was also detected by urease method using standard protocol. Among the 14 samples studied, 8 showed the presence of H. pylori with both templates A and B. Among these 8 samples only 3 showed positive for the presence of H. pylori with urease method. The present work discusses the results obtained in the detection of H. pylori in template A and B by PCR method.

Prenatal diagnostic testing for infantile and late-infantile neuronal ceroid lipofusinoses (NCL) using allele specific primer extension (ASPE) / 北京大学学报(医学版)

SUMMARY Infantile (INCL, NCL1) and late-infantile (LINCL, NCL2) neuronal ceroid lipofuscinoses have been found to result from genetic deficiency of genes CLN1 and CLN2, respectively. The application of molecular analyses can facilitate prenatal diagnosis for families affected by NCL1 or NCL2, in which the familial mutation(s) have been identified. Molecular testing with allele-specific primer extension and DNA sequencing was performed in nine pregnancies, four from two NCL1 families and five from five NCL2 families. Lysosomal enzyme activity assays were carried out as well.Four fetuses from three pregnancies in NCL1 families were found to be carriers for a mutation 451C-T in the CLN1 gene and one was normal. Prenatal testing of three NCL2 families who carried mutation R208X in the CLN2 gene showed that all fetuses were carriers. In NCL2 families who carried either mutation IVS5-1C or/and IVS5-1A two normal pregnancies were detected. Our studies indicate that DNA testing, which may provide definitive prenatal diagnosis for NCL, may be used in combination with lysosomal enzyme activity analyses.

Association of HLA-DRB1, DQB1 Alleles with Chronic Urticaria / 华中科技大学学报（医学）（英德文版）

Summary: In order to investigate the association of genotypes of HLA-DRB1 and HLA-DQB1 alleles with the genetic susceptibility of chronic urticaria (CU), genotypes of HLA-DRB1 and HLA-DQB1 genes were detected by polymerase chain reactions with sequence-specific primers (PCR-SSP) in 42 patients with CU (19 men and 23 women, mean age 30.67±12.45 y old as well as 193 racially matched healthy persons in ethnic Han from Hubei provinece. Gene frequencies of HLA-DRB1*12, *0901 (RR=3.11, χ2=7.579, P=0.006; RR=2.47, χ2=5.684, P=0.017) were significantly increased in CU patients as compared with that in healthy people. Gene frequencies of HLA-DQB1*05 (RR＝0.26, χ2=6.683, P=0.01) were significantly decreased in CU patients. It was suggested that CU was found strongly associated with HLA-DRB1*12, *0901 and HLA-DQB1*05, the former might be the genetic markers for susceptibility to CU, but the latter might play a resistive role.

Polymerase Chain Reaction-Sequence Special Primers in Detecting Gene Polymorphism of Pediatrics Diseases / 实用儿科临床杂志

Objective To establish an approach of polymerase chain reaction-sequence special primers(PCR-SSP) to perform polymorphism analysis for multiple genes simultaneously,in order to be used in clinical analysis.Methods The optimized PCR-SSP approach was used to analyze the polymorphism of the following genes:mutation of-308A/G and-238G/A in tumor necrosis factor-?((TNF-?)),-174G/C in interleukin-6(IL-6).Results Polymorphism analysis of multiple genes and many clinical samples could be simultaneously performed with one PCR program,showing clear genotype,quick and accurate genotype.Conclusion The optimized(PCR-SSP) is suitable for polymorphism analysis of the large-sample polygenic mononcleeotide point mutation,and it can be widely applied in clinical test for low cost,quickness and accuracy.

Association between disease susceptivity of chronic HBV infection and SNP G-944C in CⅡTA gene promoter Ⅳ / 第三军医大学学报

0.05). Conclusion The polymorphism of G-944C in CⅡTA gene promoter Ⅳ was associated with the susceptivity of chronic HBV infection, but was not associated with severity of diseases. The individuals with chronic HBV infection of CC genotype are of less possibility to develop chronic liver disease than those of other genotypes.

Evaluation of the NeoDin SSP(TM) HLA-DR Typing Kit / 대한진단검사의학회지

BACKGROUND: For HLA-DR typing, PCR-SSO (sequence specific oligonucleotide) kits are most commonly used in Korea. However, the PCR-SSO method generally shows more ambiguities than PCR-SSP (sequence specific primer) method in generic-level typing of HLA-DRB1 alleles. We evaluated the newly developed NeoDin SSP(TM) HLA-DR Typing kit based on the PCR-SSP method. METHODS: A total of 118 selected samples with known DRB1 alleles were tested with the NeoDin SSP(TM) HLA-DR Typing kit and the band patterns were interpreted by two investigators in a blind manner. RESULTS: Correct assignment of HLA-DRB1 alleles at a generic level was possible in 117 (99.2%)out of 118 samples tested. Only one sample carrying DRB1*1403 as a homozygote showed ambiguity: DR14 homozygote versus DR14, DR13. Some HLA-DR specificities (DR8, DR11-14) were dividied into 2-11 allelic groups and the typing results (allelic groups) were fully concordant with the known DRB1 allelic specificities. When a proper concentration of DNA with good purity was used, band patterns were clear and easy to read and no false positive or false negative band was observed in the DRB1 assignments. The occasional presence of 1-2 faint nonspecific bands did not much influence the correct assignments of specific bands. In a small proportion (5 samples, 4%) of samples tested, a random occurrence of PCR failure of 1-2 internal control bands was observed; however it did not affect the correct assignments of DRB1 alleles. CONCLUSIONS: When a proper concentration of DNA with good purity is used, the correct assignments of HLA-DRB1 alleles at a generic level are possible in >99% of the samples without ambiguity, using the NeoDin SSP kit.

Evaluation of the NeoDin SSP(TM) HLA-DR Typing Kit / 대한진단검사의학회지

BACKGROUND: For HLA-DR typing, PCR-SSO (sequence specific oligonucleotide) kits are most commonly used in Korea. However, the PCR-SSO method generally shows more ambiguities than PCR-SSP (sequence specific primer) method in generic-level typing of HLA-DRB1 alleles. We evaluated the newly developed NeoDin SSP(TM) HLA-DR Typing kit based on the PCR-SSP method. METHODS: A total of 118 selected samples with known DRB1 alleles were tested with the NeoDin SSP(TM) HLA-DR Typing kit and the band patterns were interpreted by two investigators in a blind manner. RESULTS: Correct assignment of HLA-DRB1 alleles at a generic level was possible in 117 (99.2%)out of 118 samples tested. Only one sample carrying DRB1*1403 as a homozygote showed ambiguity: DR14 homozygote versus DR14, DR13. Some HLA-DR specificities (DR8, DR11-14) were dividied into 2-11 allelic groups and the typing results (allelic groups) were fully concordant with the known DRB1 allelic specificities. When a proper concentration of DNA with good purity was used, band patterns were clear and easy to read and no false positive or false negative band was observed in the DRB1 assignments. The occasional presence of 1-2 faint nonspecific bands did not much influence the correct assignments of specific bands. In a small proportion (5 samples, 4%) of samples tested, a random occurrence of PCR failure of 1-2 internal control bands was observed; however it did not affect the correct assignments of DRB1 alleles. CONCLUSIONS: When a proper concentration of DNA with good purity is used, the correct assignments of HLA-DRB1 alleles at a generic level are possible in >99% of the samples without ambiguity, using the NeoDin SSP kit.

ITS Primers with Enhanced Specificity to Detect the Ectomycorrhizal Fungi in the Roots of Wood Plants

With universal primer ITS1-F, the specific DHJ2 primer was developed to detect the Ectomycorrhizal (ECM) root tips in soil and to identify the species of ECM fungi, as based on DNA sequences of rDNA stored in GeneBank of NCBI. This primer was designed with the common sites of rDNA of Amanita and Boletus, and was also designed with several DNA programs provided by NCBI. The DNA fragments synthesized by PCR were calculated to be 1,000 to 1,200 bps of DNA located to 18s to 28s rDNA to contain two variable sites of ITS, indicating much diversities for specific species or ecotypes of ECM fungi. The primer DHJ2 reacted with the genomic DNA's extracted from the tissues of basidiocarp at the rate of 73 of 80 fungi collected produced single bands with a 1,100 bps length. The DNA fragment synthesized with the genomic DNA that extracted from eight ECM tips of Pinus densiflora was confirmed and analysized to the rDNAs of ECM in full sequences, and informed to be a ECM fungal species in the forest.

Multiple Symbiotic Associations Found in the Roots of Botrychium ternatum

Two types of mycorrhizae, orchid (OM) and arbuscular mycorrhizae (AM), were observed in the cortical cells of Botrychium ternatum roots. The vesicles or arbuscules of AM fungi were examined and the fresh or digestive pelotons by other species of basidiomycetes were also observed in the roots under light microscope. These symbioses were, as the genomic DNAs extracted from roots of B. ternatum reacted with the specific primers, confirmed with PCR technique, being added to more strong evidences. These discoveries were rarely happened in the roots, especially a fern in nature. OM was observed in the roots of B. ternatum collected from the nationwide areas, whereas AM was only in the roots of B. ternatum collected from Chung-Buk areas. It is speculated that OM are associated with the nitrogen cycle in Islands and the growth of B. ternatum in the inland of Central Korea is related to both the phosphate and nitrogen cycle in the nature. The results suggest that B. ternatum is a typical species with two types of mycorrhizae under various growing conditions.

Molecular Detection of Phellinus linteus and P. baumii by PCR Specific Primer

Specific primer sets based on ribosomal DNA (rDNA) internal transcribed specer (ITS) sequences were designed for rapid detection of Phellinus linteus and P. baumii. Polymerase chain reaction (PCR) with these primers produced unique bands for each Phellinus species. The annealing temperature range is from 40degrees C to 55degrees C. The length of PCR products (P. linteus and P. baumii) using designed combinative primer sets of PL1F, PL2R, PB1F, PB2R, ITS5F and ITS4R, were from 520 bp to 730 bp. Fifteen strains of Phellinus species including P. linteus, P. baumii, P. weirianus, P. johnsonianus, P. rhabarberinus, P. pini, P. gilvus, P. igniarius, P. nigricans and P. laevigatus were examined in this study. Five strains, including two isolated strains of P. linteus (MPNU 7001 and MPNU 7002), and two isolated strains of P. baumii (MPNU 7004 and MPNU 7005) were shown to have about 520 bp (PL1F-PL2R), 700 bp (ITS5F-PL2R) and 600 bp (PB1F-ITS4R)-sized PCR single bands respectively. This molecular genetic technique provided a useful method for rapid detection and identification of P. linteus and P. baumii.