Paper Information

Journal:   IRANIAN JOURNAL OF PUBLIC HEALTH   AUGUST 2014 , Volume 43 , Number SUPPLEMENT 2; Page(s) 71 To 71.
 
Paper: 

DESIGN OF NOVEL INTERNAL POSITIVE CONTROL (IPC) FOR MOLECULAR DETECTION OF INFECTIOUS BRONCHITIS VIRUS (IBV) THROUGH REAL TIME RT PCR

 
 
Author(s):  MADHI ALI*, GHALYANCHILANGEROUDI ARASH, SOLEIMANI MOHAMMAD MEHDI, HOSSEINI SEYED MASOUD
 
* SHAHID BEHESHTI UINVERSITY, TEHRAN, IRAN
 
Abstract: 

Background: The aim of the present survey is to design and construct a plasmid containing recombinant 5'UTR and 3'UTR genes as positive control and internal positive control.
Methods: We constructed an IPC that can be amplified by the same primer pair as wild-type target RNA and the different site for probe. IPC was obtained by insertion of an exogenous DNA fragment into reference strain target by recombinant PCR and finally the production was colonized in pTZ57RT.Afterwards, the plasmid constructs were transformed into E. coli TOP10F ? host strain. Screening the desired recombinant clone was carried out using colony PCR.
Results: Sequencing confirmed the presence of the desire insert (IPC sequence) in recombinant plasmid. Consequently, the IPC fragment was longer than the target gene while both ends had similar attachments to the same primer pair. The probe site is different in positive control and IPC.
Conclusion: An internal control of amplification was constructed by recombinant PCR to detect PCR inhibitors. This exogenous DNA was included in the reaction mixture and amplified with the target gene. This detection was successfully applied to the diagnosis of Infectious bronchitis (IB) in clinical samples. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of RT_ -PCR and builds confidence in negative results obtained with agent specific assays.

 
Keyword(s): INFECTIOUS BRONCHITIS, IRAN
 
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