Construction and Evaluation of a Novel Internal Positive Control (IPC) for Detection of Coxiella burnetii by PCR

Abstract

Background:Due to the limitations of the classical methods to detect Coxiella burnetii, direct diagnosis of the pathogen using PCR techniques is still the preferable approach. However, false negative results owing to the presence of PCR inhibitors are troublesome.Objectives:In order to identify the inhibitors during PCR assay, an internal positive control (IPC) was designed based on 16SrRNA gene of C. burnetii.Materials and Methods:In the current study, the initial and ending parts of the target gene in an external positive control plasmid (pTZ57R/T-16S) amplified using internal primers which had a BglII restriction site on the 5´ends. Both PCR products (fragments 1 and 2) were cloned into pTZ57R/T vector. Following BglII enzyme digestion, the two obtained linear plasmids were ligated. The ligation product was transformed into Escherichia coli Top10 Fʹ. Screening of the desired recombinant clone was carried out using colony PCR.Results:The size of the PCR product was equal to the sum of the first and second fragments. 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.Conclusions:The results showed that direct fusion of the recombinant plasmids containing the initial and ending parts of the target gene are simple and cost-effective techniques for increasing the length of the fragment and constructing IPC.

Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces

“…However, PCR may yield false negative results when applied to complex matrices due to the presence of substances such as heme, bilirubins, bile salts, and complex carbohydrates [13] that inhibit the amplification reaction [12]. False negative results in PCR can be attributed to expired reagents, poor techniques, or equipment failure [18,25]; therefore, the introduction of an internal amplification control (IAC) in the PCR reaction mix is mandatory [14,15,21].…”