STANDARDIZATION OF DIAGNOSTIC PCR BY INTERNAL AMPLIFICATION CONTROL

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SHAHHOSSEINY MOHAMMAD HASSAN; GHAHRI MARYAM; MAHMOUDI MOHAMMAD AMIN; MOSLEMI ELHAM

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Journal: Journal of Microbial Biology Year:2015 | Volume:4 | Issue:15 Page(s): 55-68

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Title

STANDARDIZATION OF DIAGNOSTIC PCR BY INTERNAL AMPLIFICATION CONTROL

Author(s)

SHAHHOSSEINY MOHAMMAD HASSAN | GHAHRI MARYAM | MAHMOUDI MOHAMMAD AMIN | MOSLEMI ELHAM | Issue Writer Certificate

Keywords

PCRQ2

INTERNAL CONTROLQ2

PCR- CLONINGQ2

Abstract

Introduction: Different data resulting from non-standard molecular techniques in laboratories were considered as one of the deficiencies about amplification techniques. One of the most important aspects about the articles published in PCR detection until now, is absence of INTERNAL CONTROL (IC) in majority of them. The aim of this study is to design and develop a simple and rapid method to produce INTERNAL CONTROL for PCR test and its future application in the routine recognition laboratories.Materials and methods: To produce competitive INTERNAL CONTROL in this study, composite primers and PCR-Cloning method were used. We designed and synthesized the forward and reverse primers of PCR diagnostic test of Hepatitis B virus(HBV), Herpes simplex virus(HSV), Mycobacterium tuberculosis(MTB), Mycoplasma pneumonia(Mpn), Cryptococcus neoformans(Cne), Salmonella spp (Sal.sp) and Mycoplasma spp (M.sp) in the 5’ primers of Leishmania Kintoplast gene in the tail form. The amplified INTERNAL CONTROLs were attached to pTZ57R and transformed in E.coli JM107 bacteria. The minimum IC number was studied using dilution technique and also PCR response spectrum with IC.Results: The size of HBV, HSV, MTB, MPN, C.ne, Sal.sp, M.sp diagnostic product with specific primers were 262, 454, 245, 345, 415, 284, 272 bp and corresponding INTERNAL CONTROL also were 660, 662, 660, 669, 661, 668, 672 bp respectively, and desirable difference observed between PCR, IC regarding the size was easy to separate in the gel. The minimum IC was identified to 1000 in each reaction and maximum PCR test sensitivity with IC was provided for entire agents appropriately. Further, in specific test using different agents any unwanted product was not observed too.Discussion and conclusion: The negative and positive false results in PCR tests are one of the difficulties of this exacting technique which may lead to decrease its performance. Using an INTERNAL CONTROL in molecular detection method as an INTERNAL CONTROLling system, could identify these errors.

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APA: Copy

SHAHHOSSEINY, MOHAMMAD HASSAN, GHAHRI, MARYAM, MAHMOUDI, MOHAMMAD AMIN, & MOSLEMI, ELHAM. (2015). STANDARDIZATION OF DIAGNOSTIC PCR BY INTERNAL AMPLIFICATION CONTROL. BIOLOGICAL JOURNAL OF MICROORGANISM, 4(15), 55-68. SID. https://sid.ir/paper/237388/en

Vancouver: Copy

SHAHHOSSEINY MOHAMMAD HASSAN, GHAHRI MARYAM, MAHMOUDI MOHAMMAD AMIN, MOSLEMI ELHAM. STANDARDIZATION OF DIAGNOSTIC PCR BY INTERNAL AMPLIFICATION CONTROL. BIOLOGICAL JOURNAL OF MICROORGANISM[Internet]. 2015;4(15):55-68. Available from: https://sid.ir/paper/237388/en

IEEE: Copy

MOHAMMAD HASSAN SHAHHOSSEINY, MARYAM GHAHRI, MOHAMMAD AMIN MAHMOUDI, and ELHAM MOSLEMI, “STANDARDIZATION OF DIAGNOSTIC PCR BY INTERNAL AMPLIFICATION CONTROL,” BIOLOGICAL JOURNAL OF MICROORGANISM, vol. 4, no. 15, pp. 55–68, 2015, [Online]. Available: https://sid.ir/paper/237388/en

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