Phylogenetic analysis and designing new primers for molecular identification of Drosophila suzukii

The family Drosophilidae includes over 3750 species worldwide and over 2000 of these are species of Drosophila. Spotted wing drosophila (SWD), Drosophila suzukii is one of the most dangerous species in this family. The insects live on undamaged ripening fruits, using its peculiar serrated ovipositor to break the skin of fresh ripening fruits and lay eggs in it. Drosophila species are very difficult and practically impossible to detect at larval stages. The present investigation was conducted at the All-Russian Plant Quarantine Center and Agrarian and Technological Institute of RUDN University, Moscow, Russia in 20182020. The aim of this study was to investigate the method of accurate and rapid identification of D. suzukii, and to design specific primer pairs for pest identification by Real-Time PCR method. The real-time quantitative PCR is a fast, sensitive, repeatable and accurate method for quantifying gene transcript levels. In this study, we designed specific primers (4.Dsuz.FRP) for Real-Time PCR to identify D. suzukii from other relative species. Although D. suzukii is absent in the Russian Federation and has not been reported so far, the project could be a precautionary measure.

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Rapid Identification of Measles Virus Vaccine Genotype by Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.01879-16 ◽

2016 ◽

Vol 55 (3) ◽

pp. 735-743 ◽

Cited By ~ 13

Author(s):

Felicia Roy ◽

Lillian Mendoza ◽

Joanne Hiebert ◽

Rebecca J. McNall ◽

Bettina Bankamp ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Measles Virus ◽

Rapid Identification ◽

Rt Pcr ◽

Outbreak Response ◽

Reverse Transcription Pcr ◽

Pcr Method ◽

Measles Outbreaks ◽

Genotype A

ABSTRACT During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time reverse transcription-PCR (RT-PCR) method specific for genotype A measles virus (MeV) (MeVA RT-quantitative PCR [RT-qPCR]) that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche LightCycler 480 system and the Applied Biosystems (ABI) 7500 real-time PCR system. In comparison to the standard real-time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT-qPCR assay has been used successfully for measles surveillance in reference laboratories, and it could be readily deployed to national and subnational laboratories on a wide scale.

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Real-time quantitative PCR detection ofEscherichia coliO157:H7

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236207001349 ◽

2007 ◽

Vol 4 (1) ◽

pp. 15-19 ◽

Cited By ~ 4

Author(s):

Chen Si ◽

Huang Kun-Lun ◽

Xu Wen-Tao ◽

Li Yuan ◽

Luo Yun-Bo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Polymerase Chain Reaction ◽

Amplification Product ◽

Chain Reaction ◽

Standard Curve ◽

Real Time Quantitative Pcr ◽

Pcr Method ◽

Polymerase Chain ◽

Dissociation Curves

AbstractA rapid and accurate real-time quantitative polymerase chain reaction (real-time PCR) method with SYBR Green I was established for detectingEscherichia coliO157:H7. A pair of primers were designed to amplify theeaegene. The dissociation curves showed that the amplification product was very specific. The optimal conditions and standard curve were established. The result indicated that real-time PCR was 1000 times more sensitive than ordinary PCR.

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Determination of Cry1Ac copy number in transgenic pigeonpeaplants using quantitative real time PCR.

Legume Research - An International Journal ◽

10.18805/lr.v0iof.11300 ◽

2016 ◽

Author(s):

Meenakshi . Jain ◽

Surender . Khatodia ◽

Pushpa . Kharb ◽

Praveen . Batra ◽

Vijay K. Chowdhury

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Cost Effective ◽

Single Copy ◽

Quantitative Real Time Pcr ◽

Real Time Quantitative Pcr ◽

Specificity And Sensitivity ◽

Transgenic Lines ◽

Pcr Method

Copy number of Cry1Ac in transgenic pigeonpea plants was determined by quantitative real time PCR using Syber Green as fluorescence indicator. Gene specific primers designed to amplify relatively long amplicons (400 – 600 bp), for Cry1Ac was used to increase specificity and sensitivity of Real time PCR. Estimated copy number in transgenic lines using real-time quantitative PCR and southern hybridization was correlated and found to be same i.e. single copy number. This study shows effectivness of real-time PCR method for estimating the transgene copy number in transgenic pigeonpea plants by a simple, accurate and cost effective manner.Keywords: Copy number, Cry1Ac, Pigeonpea transformation, Real-time PCR, Syber green.

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Development of a real-time PCR method (Taqman) for rapid identification and quantification of Prorocentrum donghaiense

Journal of Ocean University of China ◽

10.1007/s11802-012-1911-0 ◽

2012 ◽

Vol 11 (3) ◽

pp. 366-374 ◽

Cited By ~ 1

Author(s):

Jian Yuan ◽

Tiezhu Mi ◽

Yu Zhen ◽

Zhigang Yu

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Identification ◽

Prorocentrum Donghaiense ◽

Pcr Method ◽

Identification And Quantification

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Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

Anti-Infective Agents ◽

10.2174/2211352518999200629165108 ◽

2020 ◽

Vol 18 ◽

Author(s):

Pegah Shakib ◽

Mohammad Reza Zolfaghari

Keyword(s):

Streptococcus Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Cross Sectional Study ◽

Laboratory Culture ◽

Cross Sectional ◽

Negative Results ◽

False Negative Results ◽

Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

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

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

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.

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