Development of an enterovirus specific PCR method for the quantification of enterovirus genomes in blood of diabetes patients

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.

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

ACTA MEDICA IRANICA ◽

10.18502/acta.v57i9.2641 ◽

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.

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

Letters in Applied Microbiology ◽

10.1111/lam.12818 ◽

2017 ◽

Vol 66 (1) ◽

pp. 86-92 ◽

Cited By ~ 1

Author(s):

S. Yamashita ◽

H. Nakagawa ◽

T. Sakaguchi ◽

T-H. Arima ◽

Y. Kikoku

Keyword(s):

Heat Resistant ◽

Specific Pcr ◽

Pcr Method ◽

Species Specific

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

Oncology Reports ◽

10.3892/or.6.1.219 ◽

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

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

Diagnostics ◽

10.3390/diagnostics10110872 ◽

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.

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

Plant Disease ◽

10.1094/pdis-09-18-1624-re ◽

2019 ◽

Vol 103 (8) ◽

pp. 1998-2009 ◽

Cited By ~ 4

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.

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

Plant Disease ◽

10.1094/pdis.2002.86.6.682 ◽

2002 ◽

Vol 86 (6) ◽

pp. 682-686 ◽

Cited By ~ 27

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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Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.3883-3891.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 3883-3891 ◽

Cited By ~ 101

Author(s):

Yukiko Hara-Kudo ◽

Kanji Sugiyama ◽

Mitsuaki Nishibuchi ◽

Ashrafuzzaman Chowdhury ◽

Jun Yatsuyanagi ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

The United States ◽

Most Probable Number ◽

Asian Countries ◽

Probable Number ◽

Specific Pcr ◽

Thermostable Direct Hemolysin ◽

Chromogenic Agar ◽

Pcr Method ◽

Arbitrarily Primed Pcr

ABSTRACT Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.

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