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

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 F'. 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.