HLA-DQB1?LOW-RESOLUTION? TYPING BY PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS (PCR-SSP)

Abstract PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar “Williams 82” [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca2+-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.

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Specific DNA identification of Pheretima in the Naoxintong capsule

Chinese Medicine ◽

10.1186/s13020-019-0264-7 ◽

2019 ◽

Vol 14 (1) ◽

Cited By ~ 1

Author(s):

Xiaoxiao Zhu ◽

Hoi-Yan Wu ◽

Pang-Chui Shaw ◽

Wei Peng ◽

Weiwei Su

Keyword(s):

Pcr Amplification ◽

Cerebrovascular Diseases ◽

Coi Gene ◽

Chemical Marker ◽

Dna Identification ◽

Specific Primers ◽

Blast Search ◽

Pcr Products ◽

Crude Drugs ◽

Nucleotide Database

Abstract Background Pheretima is a minister drug in Naoxintong capsule (NXTC), a well-known traditional Chinese medicine (TCM) formula for the treatment of cardiovascular and cerebrovascular diseases. Owing to the loss of morphological and microscopic characteristics and the lack of recognized chemical marker, it is difficult to identify Pheretima in NXTC. This study aims to evaluate the feasibility of using DNA techniques to authenticate Pheretima, especially when it is processed into NXTC. Methods DNA was extracted from crude drugs of the genuine and adulterant species, as well as nine batches of NXTCs. Based on mitochondrial cytochrome c oxidase subunit I (COI) gene, specific primers were designed for two genera of genuine species, Metaphire and Amynthas, respectively. PCR amplification was performed with the designed primers on crude drugs of Pheretima and NXTCs. The purified PCR products were sequenced and the obtained sequences were identified to species level with top hit of similarity with BLAST against GenBank nucleotide database. Results Primers MF2R2 and AF3R1 could amplify specific DNA fragments with sizes around 230–250 bp, both in crude drugs and NXTC. With sequencing and the BLAST search, identities of the tested samples were found. Conclusion This study indicated that the molecular approach is effective for identifying Pheretima in NXTC. Therefore, DNA identification may contribute to the quality control and assurance of NXTC.

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Automation of HLA class II genotyping by PCR amplification with sequence-specific primers

Human Immunology ◽

10.1016/0198-8859(94)90196-1 ◽

1994 ◽

Vol 39 (2) ◽

pp. 141

Author(s):

P. Merel ◽

F. Comeau ◽

B. Dupin ◽

R. Destrebecq ◽

G. Vezon

Keyword(s):

Pcr Amplification ◽

Class Ii ◽

Hla Class Ii ◽

Specific Primers

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Field and Laboratory Methods for DNA Studies on Deep-sea Isopod Crustaceans

Polish Polar Research ◽

10.2478/popore-2014-0018 ◽

2014 ◽

Vol 35 (2) ◽

pp. 203-224 ◽

Cited By ~ 34

Author(s):

Torben Riehl ◽

Nils Brenke ◽

Saskia Brix ◽

Amy Driskell ◽

Stefanie Kaiser ◽

...

Keyword(s):

Deep Sea ◽

Pcr Amplification ◽

The Other ◽

Universal Primers ◽

Success Rates ◽

Specific Primers ◽

Scientific Publications ◽

Cycle Sequencing ◽

Laboratory Methods ◽

Set Up

AbstractField and laboratory protocols that originally led to the success of published studies have previously been only briefly laid out in the methods sections of scientific publications. For the sake of repeatability, we regard the details of the methodology that allowed broad-range DNA studies on deep-sea isopods too valuable to be neglected. Here, a comprehensive summary of protocols for the retrieval of the samples, fixation on board research vessels, PCR amplification and cycle sequencing of altogether six loci (three mitochondrial and three nuclear) is provided. These were adapted from previous protocols and developed especially for asellote Isopoda from deep-sea samples but have been successfully used in some other peracarids as well. In total, about 2300 specimens of isopods, 100 amphipods and 300 tanaids were sequenced mainly for COI and 16S and partly for the other markers. Although we did not set up an experimental design, we were able to analyze amplification and sequencing success of different methods on 16S and compare success rates for COI and 16S. The primer pair 16S SF/SR was generally reliable and led to better results than universal primers in all studied Janiroidea, except Munnopsidae and Dendrotionidae. The widely applied universal primers for the barcoding region of COI are problematic to use in deep-sea isopods with a success rate of 45–79% varying with family. To improve this, we recommend the development of taxon-specific primers.

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PCR-based molecular discrimination betweenMaritrema eroliaeandProbolocoryphe uca(Digenea: Microphallidae) in Kuwait Bay

Journal of Helminthology ◽

10.1017/s0022149x12000892 ◽

2013 ◽

Vol 88 (2) ◽

pp. 177-182

Author(s):

W.Y. Al-Kandari ◽

S.A. Al-Bustan ◽

M. Alnaqeeb ◽

A.M. Isaac

Keyword(s):

Fragment Length Polymorphism ◽

Pcr Amplification ◽

Direct Pcr ◽

Specific Primers ◽

Kuwait Bay ◽

Future Studies ◽

Larval Stages ◽

Marine Snails ◽

Pcr Rflp ◽

Species Specific

AbstractMicrophallid trematodes are common parasites in marine snails and crustacean hosts at Kuwait Bay. The larval stages of two microphallids,Maritrema eroliaeandProbolocoryphe uca, are difficult to differentiate morphologically. In this study, two PCR-based techniques were established for quick and accurate discrimination between the larval stages of the two microphallid species, employing restriction fragment length polymorphism (PCR-RFLP) and species-specific primers. Both techniques utilized nucleotide differences in the second internal transcribed region (ITS2) of the ribosomal DNA (rDNA) in the two species. For the PCR-RFLP technique, restriction enzymeAvaII was selected and it generated different restriction profiles among the two microphallids. In addition, species-specific primers were prepared for each microphallid species that amplified distinctive fragments. Both techniques showed that the larval stages of the two microphallid species can be identified accurately. However, direct PCR amplification using species-specific primers was more advantageous than the PCR-RFLP technique since it allowed rapid and specific discrimination between the two species. This technique provides a useful tool that can be used in future studies for the study of the distribution of microphallid species and their definitive hosts at different localities of Kuwait Bay.

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The condundrum of feline trichomonosis – The more we learn the ‘trickier’ it gets

Journal of Feline Medicine and Surgery ◽

10.1177/1098612x17693499 ◽

2017 ◽

Vol 19 (3) ◽

pp. 261-274 ◽

Cited By ~ 16

Author(s):

Jody L Gookin ◽

Katherine Hanrahan ◽

Michael G Levy

Keyword(s):

Chronic Diarrhea ◽

Safety Margin ◽

Pcr Amplification ◽

Evidence Base ◽

Domestic Cat ◽

Tritrichomonas Foetus ◽

Specific Primers ◽

Clinical Resistance ◽

Comprehensive Examination ◽

Species Specific

Practical relevance: Trichomonosis of the large intestine of the cat was described as a cause of chronic diarrhea over 20 years ago. The trichomonad was identified as Tritrichomonas foetus, with a genotype that is distinct from venereal T foetus of cattle. Clinical challenges: Despite multiple means for diagnosis of the infection, including light microscopy, protozoal culture and PCR amplification using species-specific primers, tests with even greater sensitivity are needed. Feline trichomonosis is resistant to all commonly used antiprotozoal drugs. Ronidazole is currently the only drug demonstrated to be effective in eliminating the infection from cats; however, this drug has a narrow safety margin and clinical resistance is increasingly recognized. The more we learn about trichomonosis in cats, the more complicated and controversial the infection has become, ranging from what we should call the organism to whether we should even bother trying to treat it. Global importance: Feline trichomonosis is recognized to occur worldwide and is regarded as one of the most common infectious causes of colitis in the domestic cat. The infection is widespread in catteries and shelters; and, while remission of diarrhea may occur over time, persistence of the infection is common. Evidence base: This review provides a comprehensive examination of what is currently known about feline trichomonosis and pinpoints areas, based on the authors’ opinion, where further research is needed.

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Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers

Plant Disease ◽

10.1094/pdis.1997.81.10.1155 ◽

1997 ◽

Vol 81 (10) ◽

pp. 1155-1160 ◽

Cited By ~ 49

Author(s):

K. Kageyama ◽

A. Ohyama ◽

M. Hyakumachi

Keyword(s):

Polymerase Chain Reaction ◽

Internal Transcribed Spacer ◽

Pcr Amplification ◽

Its Region ◽

Pythium Ultimum ◽

Pythium Species ◽

Chain Reaction ◽

Specific Primers ◽

Polymerase Chain ◽

Species Specific

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

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