Causes of HLA-A2 reactive antibodies and analysis of their eplets / 中国输血杂志

【Objective】 To analyze differences of eplets between the patient who generated HLA allele-specific antibodies after platelet transfusion with donors. 【Methods】 The HLA genotypes of the patient and donors were detected by PCR-SBT, and the Luminex single antigen beads coating was used to screen HLA-Ⅰ antibodies in the patient’s serum. HLA Matchmaker was utilized to analyze different amino acids and eplets. 【Results】 The patient carried HLA-A*02∶03 allele, and HLA-A2 antibodies were found in his serum after platelet transfusion (A*02∶01, A*02∶06, and A*02∶07). Sequence alignment showed that the patient′s A*02∶03 has a difference in position 149, which resulted in a different eplet between A*02∶03 and A*02∶01, A*02∶06, A*02∶07 and then induced the production of antibodies. 【Conclusion】 HLA antibodies are specific for HLA epitopes that have structural differences due to amino acid differences between HLA alleles, suggesting that high-resolution typing of HLA-A, -B need to be conducted in patients and donors, and the acceptable mismatch of HLA should be determined based on epitopes rather than antigens, so as to reduce alloimmune response and improve platelet count after transfusion.

Analysis of HLA allele-specific antibodies and Eplets in patients with platelet transfusion refractoriness / 中国输血杂志

【Objective】 To analyze the specificity and Eplets of HLA allele-specific antibody in patients with platelet transfusion refractoriness (PTR). 【Methods】 HLA-A and B genotypes were detected by PCR-SBT, and HLA-Ⅰ antibodies in patients′ serum were detected by Luminex single antigen beads coating method. IPD-IMGT/HLA Database was used to find the differential amino acids of allele-specific antibodies, and HLA Eplet database was used to analyze the registry Eplets. 【Results】 HLA allele-specific antibodies were found in 12 out of 82 patients with PTR.After sequence alignment, a total of 18 differential amino acids were found, such as 19E>19K, 166D>116E, 167G>167W and so on. Among these differential amino acids, 16 registry Eplets were retrieved such as 19E>19K, 95I>95L, 113YD>113HD and so on.The amino acid substitution of 166DG>166EW, 70Q>70H, 67S>67Y, 94I>94T, 82LR>82RG, and 211G>211A may form new Eplets that have not been registered.The antigens of A11, A24, B15, B27 and B38 can be further subdivided into HLA narrow specific antigens. 【Conclusion】 It was found that there were HLA allele-specific antibodies in patients with PTR, suggesting that high-resolution typing of HLA-A, B should be carried out for these patients and platelet donors in HLA compatible transfusion of PTR.

Analysis and prospect of platelet compatible transfusion / 中国输血杂志

Platelet compatible transfusion can effectively solve the immune mediated platelet transfusion refractoriness (PTR), save platelet resources and improve blood safety. This paper comments and prospects the compatibility modes of HLA, HPA and CD36, HLA antibody titer, antigen immunogenicity and the development of platelet compatible transfusion. The pattern of HLA compatible platelets involves the matching in the alleles, antigens and epitopes levels, respectively, as well as avoidance donor specificity antibody (DSA) method. While setting the mean fluorescence intensity (MFI) threshold of avoidance DSA needs to be explored when using the DSA prediction method. Allele specific HLA antibodies can be found in the patients with PTR. Therefore, the patients and donors should be genotyped for HLA-A, -B loci at high-resolution level in order to avoid allele specific HLA antibodies. The immunogenicity of various antigens or epitopes at HLA-A and -B loci are different. Selecting donor platelets with low antigen expression or low immunogenicity may be a way of HLA compatible platelets. As the probability and type of HPA antibody production are different in the various populations, the approaching of compatibility HPA involves allele matching and avoidance DSA. As to CD36, the compatibility mode mainly refers to avoidance DSA, which means blood donors with CD36 antigen type Ⅰdeficiency are preferentially selected, and then those with CD36 antigen type Ⅱ deficiency. In the future, more attention should be paid to the scale up of database capacity and update of the information construction. The time waiting for compatible platelets transfusion in clinical could be significantly shortened if the requiring and matching are only conducted within the inventory and candidate platelets.

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.

Genetic study in Aedes (Stegomyia) aegypti (Linnaeus, 1762) from Londrina (Paraná State, Brazil): an approach to population structure and pyrethroid resistance

Abstract Londrina is the fourth most populous city in southern Brazil. Its subtropical weather with rain in all seasons, as well as its high population density, make the city perfect for the Aedes aegypti (Linnaeus, 1762) life cycle. Over the last few years, Londrina presented high infestation indexes and was one of the cities with the most reported cases of dengue. Uncontrolled use of synthetic insecticides may influence the mosquito's genetic composition. In this paper, we studied mitochondrial DNA and kdr mutations in Aedes aegypti. The analysis of the ND4 gene in 330 specimens showed the presence of 27 haplotypes. The pyrethroid resistance alleles (kdr) evaluated are present in the collected populations, with a 50% frequency of the Val1016Ile and 48% of the Phe1534Cys mutations. Such analysis of the mutations in the populations collected at the State University of Londrina's campus - a microenvironment that differs from the rest of the city - showed frequencies of 57% and 62%, respectively. The low gene flow observed, Nm = 0.11 and Nm = 0.10, along with the elevated differentiation, Fst = 0.19 and Fst = 0.18, among populations suggest an influence of genetic drift. The strong presence of resistance alleles kdr in the city is evident, which demonstrates that even with the interruption of the use of pyrethroids by the National Dengue Control Program, resistance may be maintained due to domestic use. Thus, the results have shown the need for genetic monitoring, alongside other entomological surveillance monitoring tools, to create strategies of mosquito control.

Rapid quality detection system of Lonicerae Japonicae Flos formula granules / 中国中药杂志

The application of traditional Chinese medicine(TCM) formula granules in clinical practice is gradually extensive. However, TCM formula granules is still lacking rapid and simple quality control standards. In this study, allele-specific PCR and enzyme-linked immunoassay(ELISA) was used for rapid detection of the quality of Lonicerae Japonicae Flos formula granules. The authenticity of Lonicerae Japonicae Flos formula granules was identified by allele-specific PCR and index component was detected by ELISA. Thus, it lays a foundation for the establishment of rapid quality detection standard for Lonicerae Japonicae Flos formula granules, and also provides reference for other studies on the quality standard of traditional Chinese medicine formula granules.

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.

Development of KASP marker for cytoplasmic male sterility in Nicotiana tabacum and utilization in trait introgression

Cytoplasmic male sterility (CMS) is an important trait for large-scale hybrid seed production which avoids manual emasculation and undesired horizontal spread of pollen.Rearrangements in mitochondrial genome in terms of deletions and insertions are frequent causes leading to CMS. Mitochondrial ATP synthase is a multisubunit molecular machine which is involved in synthesis of ATP. In this study, three mutations in ATPase subunit 6 were identified and their cosegregation with male sterility was established using tobacco male sterile hybrids and Nicotiana suvaolensis. A breeder friendly Kompetitive allele specific polymerase chain reaction (KASP) SNP marker was developed for high throughput and quick genotyping. Introgression of this trait into selected germplasm lines (n = 9) was achieved based on foreground for CMS and background selection for recurrent parent using KASP marker and 50K custom tobacco SNP array, respectively. Analysis of genotyping data from SNP array revealed the presence of 88–99% of recurrent parent genome in BC3F1 plants. The selected BC3F1 plants exhibit CMS and are indistinguishable from the fertile recurrent parent (germplasm) in terms of plant morphology.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay

Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. Methods: Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST). Results: The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR). Interpretation & conclusions: The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

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.

Identification of Fallopia multiflora by allele-specific polymerase chain reaction / 中草药

Objective :To establish a rapid molecular identification method for Fallopia multiflora and its adulterants. Methods: Based on psbA-trnH sequences of F. multiflora and its adulterants, the SNP site was searched and the specific primers were designed. The allele-specific PCR amplification of F. multiflora and its adulterants from different producing areas was carried out and the reaction system was optimized. Results: When the annealing temperature was raised to 48 ℃ with 30 cycle number, only the template DNA of F. multiflora could be amplified to obtain the specific 191 bp band whereas the diagnostic PCRs of the other adulterants were all negative. Conclusion: It’s simple and reliable to identify the authenticity of F. multifloa by allele loci specific PCR.

Evaluation of High Resolution Melting Technique for Detection of JAK2-V617F Mutation in Formalin-Fixed Paraffin-Embedded Specimen from Myeloproliferative Neoplasm Cases

@#Introduction: Myeloproliferative neoplasm (MPN) is a group of myeloid disorders which leads to erythrocytosis, thrombocytosis and leucocytosis. MPN with BCR-ABL positive is chronic myeloid leukaemia (CML) while BCR-ABL negative MPN includes polycythaemia Vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and MF. Beside peripheral blood specimen, formalin-fixed paraffin-embedded (FFPE) marrow specimen can be used for detection of this mutation. Unfortunately, FFPE produces low quality DNA that put a challenge for successful amplification of DNA. We aimed to evaluate the utility of High Resolution Melting (HRM) analysis for detection of JAK2-V617F mutation in FFPE specimen from MPN cases. Materials and Methods: This study is a descriptive crosssectional study. Forty FFPE marrow specimens were retrieved from the years 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR was done for validation of results. Positive samples were subjected to Sanger sequencing. Results: JAK2-V617F mutation was positive in 13 out of 40 MPN cases. Level of agreement between HRM and AS-PCR was 97.5%. Conclusion: HRM is a rapid and powerful diagnostic assay which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimen.

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 Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR / 中国实验方剂学杂志

Objective: To obtain a rapid,efficiency and convenient polymerase Chain reaction(PCR) identification method for medicinal Cervi Cornu Pantotrichum,Cervi Cornu and its common adulterates. Method: Based on three single nucleotide polymorphisms (SNP) of Cytb gene DNA sequences among Cervus nippon,C. elaphus and its adulterants,a pair of species-specific primers (LR-238.F and LR-238.R) was designed,the reaction conditions were optimized,and the PCR method for identification was explored and verified in terms of tolerance and feasibility. Result: Through the established allele-specific PCR method,under the annealing temperature of 56℃ and cycle number of 35,250 bp of fragments were amplified from DNA templates of Cervi Cornu Pantotrichum,Cervi Cornu and its subspecies in origin animal samples as well as herbal medicines. All of the adulterants species of Przewalskium albirostris,Cervus eldi,Odocoileus hemionus,Dama dama,Alces alces,Elaphurus davidianus,Capreolus pygargus,Rusa unicolor and Rangifer tarandus were negative by the PCR assay. Conclusion: The identification primer is highly specific,and the allele-specific PCR identification method established in this paper can accurately identify the medicinal Cervi Cornu Pantotrichum and Cervi Cornu.

Identification of uighur medicine Cichorium glandulosum and its adulterants by Allele-Specific PCR / 中草药

Objective To establish molecular identification method of Cichorium glandulosum and its adulterants Cichorium intybus by Allele-Specific PCR. Methods The samples of C. glandulosum and C. intybus were collected in different geographical areas. The DNA was extracted, and rbcL gene segments were amplified and sequenced directionally. The multiple sequences were aligned by using Clustal W. Specific primers were designed and amplified according to its variable sites, and PCR reaction system was optimized to determine detection limits and establish Allele-Specific PCR identification method. Results According to Allele-Specific PCR system established in this study for C. glandulosum, the optimization results was a total of 30 μL reaction system containing TaqDNA polymerase 0.25 μL, 10 × buffer 2.5 μL, dNTP 2.0 μL, primer 0.5 μL, template DNA 2 μL, and ddH2O 22.25 μL. The most suitable PCR amplification procedure is one cycle of predegeneration at 94 ℃ for 3 min; 32 cycles of denaturing at 94 ℃ for 30 s, annealing at the primer temperature 55 ℃ for 30 s and extending at 72 ℃ for 1 min, and extending at 72 ℃ for 7min. Through the detection of 20 medicinal materials of C. glandulosum and C. intybus, the result showed that 230 bp amplified band of target fragment was identified for C. glandulosum but no amplified band was observed for its adulterants. Conclusion In this study, we established and optimized the Allele-Specific PCR identification technology of C. glandulosum and its adulterants C. intybus, which can accurately, reliably, and effectively identify these two medicinal materials.

Allele-specific PCR to identification of Achyranthis Bidentatae Radix and Cyathulae Radix formula granules / 中国中药杂志

To establish a robust and accuracy molecular method to identify Achyranthis Bidentatae Radix and Cyathulae Radix formula granules. ITS sequences of Achyranthes bidentata and Cyathula officinalis were aligned, specific SNPs (single nucleotide polymorphisms) were excavated, specific primers were designed and allele-specific PCR method was established. The genomic DNA was successfully extracted from the herbal medicine and its formula granules by using an improved CTAB (cetyltrimethyl ammonium bromide) method and then performed PCR with the designed primers. The 187 bp specific band could be amplified only in the presentation of C. officinalis and its granules when use of C. officinalis specific primers, whereas the 162 bp band could be amplified only in the presentation of A. bidentata and its granules when use of A. bidentata specific primers. This method was also successfully applied in the identification of commercial formula granules.

Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

Background: Cultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by theΔ12 fatty acid desaturase (FAD) encoded byAhFAD2AandAhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C→A:T) ofAhFAD2Aand an "A" insertion ofAhFAD2Bresulted in high-oleic acid phenotype. Detection ofAhFAD2mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detectAhFAD2genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detectionAhFAD2genotype of large number of breeding materials. Results: Here, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC4F2seeds was aabb. Conclusions: Due to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable fordeterminingAhFAD2genotype than other methods.