scholarly journals Haplotype-specific PCR for NAT2 diplotyping

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Nuanjun Wichukchinda ◽  
Jirapa Pakdee ◽  
Punna Kunhapan ◽  
Wimala Imunchot ◽  
Licht Toyo-oka ◽  
...  
Keyword(s):  
Dose Adjustment ◽  
Indirect Method ◽  
Haplotype Inference ◽  
Tuberculosis Treatment ◽  
Antituberculosis Drug ◽  
Slow Acetylator ◽  
Specific Pcr ◽  
Sequencing Method ◽  
Pcr Method ◽  
Rapid Acetylator

AbstractN-acetyltransferase 2 (NAT2) is an enzyme that acetylates many kinds of drugs, including the antituberculosis drug isoniazid. The NAT2 gene is highly diverse across populations. An individual can be classified as having a slow acetylator (SA), an intermediate acetylator (IA), or a rapid acetylator (RA) phenotype based on its two haplotypes (diplotype) of NAT2. SA individuals are at a higher risk for isoniazid-induced hepatitis, while the RA phenotype contributes to failure in tuberculosis treatment. Being able to predict individual NAT2 phenotypes is important for dose adjustment of isoniazid. NAT2 haplotypes are commonly determined via an indirect method of statistical haplotype inference from SNP genotyping. Here, we report a direct NAT2 haplotyping method using haplotype-specific PCR (HS-PCR) for the 6 most commonly found NAT2 haplotypes: NAT2*4, NAT2*5B, NAT2*6A, NAT2*7B, NAT2*12A, and NAT2*13A. Validation of this HS-PCR method via comparison with a sequencing method in 650 Thai DNA samples (107 RA, 279 IA, and 264 SA samples) showed a concordance rate for diplotype calls of 99.23% (645/650 samples). The discordant results in 5 samples were due to 3 rare NAT2 haplotypes: NAT*5C (n = 3), NAT2*7C (n = 1), and NAT2*11A (n = 1). This novel HS-PCR method allows direct NAT2 diplotyping, enabling the implementation of NAT2 acetylator phenotypes in clinical pharmacogenetic testing.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

scholarly journals Clinical utility of the HCV drug resistance assay using the direct sequencing method and the cycleave PCR method in the dual therapy with daclatasvir and asunaprevir

Kanzo ◽  
2015 ◽  
Vol 56 (10) ◽  
pp. 533-535 ◽  
Author(s):  
Hideyuki Kudoh ◽  
Yoko Nagasawa ◽  
Michiru Ito ◽  
Nobuko Watanabe ◽  
Isao Naruse ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Utility ◽  
Direct Sequencing ◽  
Dual Therapy ◽  
Sequencing Method ◽  
Direct Sequencing Method ◽  
Pcr Method

scholarly journals Prevalence and Serotype Determination of Streptococcus agalactiae Isolated From Non-Pregnant Women in Tehran, Iran

2020 ◽  
Author(s):  
Leila Goudarzi ◽  
Mohammad Bagher Khalili ◽  
Mahmood Vakili ◽  
Maryam Sadeh
Keyword(s):  
Pregnant Women ◽  
Streptococcus Agalactiae ◽  
Group B Streptococcus ◽  
Cross Sectional Study ◽  
Chi Square ◽  
Cross Sectional ◽  
Specific Pcr ◽  
Pcr Method ◽  
Group B ◽  
Tehran Area

Consequence of Streptococcus agalactiae, Group B Streptococcus (GBS) relating infant’s diseases are well documented. Although many women carry this bacterium in their vagina, they may transfer to their infant during delivery and may result in different neonatal invasive diseases. The aim of this study was to determine the prevalence of GBS and serotyping the isolated species among un-selective non-pregnant women who attended two gynecology clinics in Tehran. In this cross-sectional study, a total of 560 vaginal samples collected from non-pregnant women. Following inoculation of the specimen on Blood Agar, the standard technology was applied for the final identification of GBS. Detected GBS species were further confirmed using specific PCR directed on dlts gene. Capsular serotyping was done by using the multiplex PCR method. The chi-square method was used for statistical analysis. Fifty (8.9%) out of 560 non-pregnant women were carriers of GBS. The most common types were III (36%), followed by type II (32%), Ia (26%), and Ib (6%), respectively. Results represent that the prevalence rate of GBS in non-pregnant women was reliable and similar to what obtained from pregnant women. In addition, the serotype III was found the most dominant types, as well as other investigations in the Tehran area. Therefore, vaccine designation based on type III is recommended.

scholarly journals Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus

2017 ◽  
Vol 66 (1) ◽  
pp. 86-92 ◽  
Author(s):  
S. Yamashita ◽  
H. Nakagawa ◽  
T. Sakaguchi ◽  
T-H. Arima ◽  
Y. Kikoku
Keyword(s):  
Heat Resistant ◽  
Specific Pcr ◽  
Pcr Method ◽  
Species Specific

Alternative exon-specific PCR method for the analysis of human CD44 isoform expression.

1999 ◽  
Author(s):  
M S Lockhart ◽  
M J Gravisaco ◽  
C Mongini ◽  
C Waldner ◽  
E Alvarez ◽  
...  
Keyword(s):  
Alternative Exon ◽  
Specific Pcr ◽  
Pcr Method ◽  
Isoform Expression

scholarly journals N-Acetyltransferase 2 Genotypes among Zulu-Speaking South Africans and Isoniazid and N-Acetyl-Isoniazid Pharmacokinetics during Antituberculosis Treatment

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Thuli Mthiyane ◽  
James Millard ◽  
John Adamson ◽  
Yusentha Balakrishna ◽  
Cathy Connolly ◽  
...  
Keyword(s):  
Nucleotide Polymorphisms ◽  
Time Curve ◽  
Slow Acetylator ◽  
South Africans ◽  
Antituberculosis Treatment ◽  
High Prevalence ◽  
Acetylator Genotype ◽  
The Difference ◽  
Median Area ◽  
Rapid Acetylator

ABSTRACT The distribution of N-acetyltransferase 2 gene (NAT2) polymorphisms varies considerably among different ethnic groups. Information on NAT2 single-nucleotide polymorphisms in the South African population is limited. We investigated NAT2 polymorphisms and their effect on isoniazid pharmacokinetics (PK) in Zulu black HIV-infected South Africans in Durban, South Africa. HIV-infected participants with culture-confirmed pulmonary tuberculosis (TB) were enrolled from two unrelated studies. Participants with culture-confirmed pulmonary TB were genotyped for the NAT2 polymorphisms 282C>T, 341T>C, 481C>T, 857G>A, 590G>A, and 803A>G using Life Technologies prevalidated TaqMan assays (Life Technologies, Paisley, UK). Participants underwent sampling for determination of plasma isoniazid and N-acetyl-isoniazid concentrations. Among the 120 patients, 63/120 (52.5%) were slow metabolizers (NAT2*5/*5), 43/120 (35.8%) had an intermediate metabolism genotype (NAT2*5/12), and 12/120 (11.7%) had a rapid metabolism genotype (NAT2*4/*11, NAT2*11/12, and NAT2*12/12). The NAT2 alleles evaluated in this study were *4, *5C, *5D, *5E, *5J, *5K, *5KA, *5T, *11A, *12A/12C, and *12M. NAT2*5 was the most frequent allele (70.4%), followed by NAT2*12 (27.9%). Fifty-eight of 60 participants in study 1 had PK results. The median area under the concentration-time curve from 0 to infinity (AUC0–∞) was 5.53 (interquartile range [IQR], 3.63 to 9.12 μg h/ml), and the maximum concentration (Cmax) was 1.47 μg/ml (IQR, 1.14 to 1.89 μg/ml). Thirty-four of 40 participants in study 2 had both PK results and NAT2 genotyping results. The median AUC0–∞ was 10.76 μg·h/ml (IQR, 8.24 to 28.96 μg·h/ml), and the Cmax was 3.14 μg/ml (IQR, 2.39 to 4.34 μg/ml). Individual polymorphisms were not equally distributed, with some being represented in small numbers. The genotype did not correlate with the phenotype, with those with a rapid acetylator genotype showing higher AUC0–∞ values than those with a slow acetylator genotype, but the difference was not significant (P = 0.43). There was a high prevalence of slow acetylator genotypes, followed by intermediate and then rapid acetylator genotypes. The poor concordance between genotype and phenotype suggests that other factors or genetic loci influence isoniazid metabolism, and these warrant further investigation in this population.

scholarly journals Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 872
Author(s):  
Alexey S. Chubarov ◽  
Igor P. Oscorbin ◽  
Maxim L. Filipenko ◽  
Alexander A. Lomzov ◽  
Dmitrii V. Pyshnyi
Keyword(s):  
Kras Mutation ◽  
Mutation Detection ◽  
Synergetic Effect ◽  
Model Systems ◽  
Wild Type ◽  
Kras Mutations ◽  
Specific Pcr ◽  
Mutational Status ◽  
Allele Specific ◽  
Pcr Method

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

scholarly journals Detection and Characterization of Fusarium oxysporum f. sp. vasinfectum VCG0114 (Race 4) Isolates of Diverse Geographic Origins

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 1998-2009 ◽  
Author(s):  
Alois A. Bell ◽  
Aixing Gu ◽  
Jim Olvey ◽  
Tanya A. Wagner ◽  
Javlon J. Tashpulatov ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Historical Collections ◽  
Specific Pcr ◽  
Reference Isolate ◽  
Compatibility Analysis ◽  
Geographic Origins ◽  
Soil Infestation ◽  
Pcr Method ◽  
Cotton Wilt ◽  
Race 4

A highly virulent cotton wilt pathogen, Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) was found in West Texas in 2017, after being known in California since 2001. Isolates obtained from wilted plants collected in 2017 from Texas, in 2015 from China, and during 2001 to 2014 from California and isolates from historical collections including the race 4 reference isolate were characterized by soil-infestation pathogenicity assays, DNA sequence analysis, and vegetative compatibility analysis. All obtained F. oxysporum f. sp. vasinfectum isolates belonged to VCG0114. All of these isolates, except one isolate from China, caused disease in a soil-infestation assay without nematodes. Thus, they belong to the nematode-independent pathotype. Texas isolates were significantly more virulent than were isolates from China or California on Gossypium barbadense ‘Pima S-7’. Four different genotypes (N, T, MT, and MiT) were identified based on the transposable element Tfo1 insertion into the PHO gene and independent MULE or MITE insertions into the Tfo1 transposon. Some significant differences in virulence were detected among the genotypes in some locations. No differences in pathogenicity were observed between the California and China collection isolates on Pima S-7, and the virulence of the major genotypes was similar on the Gossypium hirsutum cultivar ‘Stoneville 474’ or the Barbren 713 germplasm line. Simple polymerase chain reaction (PCR) methods were developed to specifically determine and detect the four genotypes within VCG0114. A specific PCR method to detect all VCG0114 isolates was also developed. These methods will facilitate the timely identification of infested fields and seed lots and the elucidation of evolutionary relationships among the isolates. This should help to closely monitor the movement of the pathogen and reduce dissemination of these devastating pathogens.

scholarly journals PCR Detection and RFLP Differentiation of Botrytis Species Associated with Neck Rot of Onion

Plant Disease ◽  
2002 ◽  
Vol 86 (6) ◽  
pp. 682-686 ◽  
Author(s):  
Karsten Nielsen ◽  
David S. Yohalem ◽  
Dan Funck Jensen
Keyword(s):  
Pcr Amplification ◽  
Pcr Detection ◽  
Sequence Characterized Amplified Region ◽  
Specific Pcr ◽  
Isolation Techniques ◽  
Detection And Identification ◽  
Fungal Dna ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Direct Amplification

Botrytis aclada and other Botrytis spp. can cause neck rot on onions, a storage disease that normally is very difficult to detect at harvest using traditional isolation techniques. Sequence characterized amplified region primers (BA2f/BA1r) were designed based on a previously cloned and amplified DNA fragment for direct amplification of isolates of Botrytis spp. associated with neck rot of onions. Digestion of the polymerase chain reaction (PCR) amplification product with the restriction enzyme ApoI makes it possible to distinguish the five groups: Botrytis aclada types AI and AII (B. allii); B. byssoidea; B. squamosa; and B. cinerea. The detection limit was 1 to 10 pg of pure fungal DNA. It was possible to detect B. aclada with the PCR method in artificially inoculated onion bulb tissue and in mature onion leaves showing no symptoms of the disease. The availability of a sensitive and specific PCR detection and identification method for Botrytis onion neck rot pathogens should facilitate ecological studies of this group of onion pathogens.

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