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Paper Information

Journal:   IRANIAN JOURNAL OF PUBLIC HEALTH   AUGUST 2014 , Volume 43 , Number SUPPLEMENT 2; Page(s) 67 To 67.
 
Paper: 

OPTIMIZATION OF REAL TIME PCR FOR DETECTION OF BRUCELL ABORTUS

 
 
Author(s):  ALAMIAN SAEED*, ZAHRAEI SALEHI TAGHI, AGHAIIPOOR KHOSRO, ESMAELIZAD MAJID, NAYERI FASAII BAHAR, AETEMADY AFSHAR, BAGHERI NEJAD RAMIN
 
* BRUCELLOSIS DEPARTMENT, RAZI VACCINE AND SERUM RESEARCH INSTITUTE KARAJ, IRAN
 
Abstract: 

Background: Brucellosis is a zoonotic disease transferred from infected animal to human. Six classic species involved B. abortus, B. melitensis, B. suis, B.canis, B.neotoma and B. ovis. In recent years four new species consist of B.ceti, B. maris, B. pinipedialis and B.inopinata were reported. Standard and classic methods are time consuming and have low specificity and sensitivity.Several Molcular methods have been developed but many of them are not able to detect all Brucella intraspecies biovars, thus developing fast and specific techniques for identification of brucellosis is essential.
Methods: Bases on bioinformatic study, B. abortus specific positions were found then a Real time PCR assay was designed. Bacterial samples used in this study consist of Brucella standard, vaccinal and 15 filed strains isolated from cow milk. High Pure PCR Template preparation Kit was used for DNA extraction. Sensitivity and specificity assays were performed.
Results: Real time PCR was 100% identical with conventional bacteriological method and phage typing assay. Moreover, based on sensitivity testes this presented real time PCR method had detection limit 400 fg B. abortus strain 544 template DNA.
Conclusion: Brucellosis detection by classic methods consists of bacterial isolation and serological assays that have sensitivity between 15 to 70 percent. Real time PCR is a fast and sensitive method for detection of brucellosis. In recent years, several studies for detection of B. abortus were reported. According to update, GenBank database used primer was not able to cover all B. abortus biovars. In this study B. abortus specific primers were designed and the study indicate real time PCR and phage typing assay have 100% identical results. In addition, real time PCR has limit of detection 400 fg template DNA, thus we suggest this presented method could be used in Brucella epidemiological and surveillance studies.

 
Keyword(s): BRUCELLA, DETECTION, REAL TIME PCR
 
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