Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR).

The research article is focused on developing a technique to improve the detection of contagious equine metritis (CEM), a sexually transmitted disease in horses, by constructing a recombinant plasmid known as CEM-POS that serves as a reaction control in Polymerase Chain Reaction (PCR) tests to reduce false positives.

Study Background

• The research is centered around Contagious Equine Metritis (CEM), a sexually transmitted bacterial disease in horses caused by Taylorella equigenitalis.
• CEM-PCR, a semi-nested PCR method, has been the primary technique for the detection of this bacterium. But this method carries the risk of producing false positives and negatives.

Study Objective

• In response to the above drawbacks, the researchers aimed to construct a recombinant plasmid, named CEM-POS, which would serve as reaction control in routine CEM-PCR examinations.
• The purpose of the CEM-POS is to ensure an adequate PCR reaction and to prevent false positives due to contamination in routine CEM-PCR tests.

Study Method

• The recombinant plasmid CEM-POS was engineered by inserting fragments from Rhodococcus equi into another recombinant plasmid named CEM-1P.
• The CEM-1P includes a T. equigenitalis-specific sequence region, creating a precise region for the detection of these specific bacteria.

Findings

• In CEM-PCR procedures, the size of the PCR product resulting from the CEM-POS plasmid had a clearly distinct size from the true positive PCR product, aiding in accurate detection.
• The constructed plasmid was noted to retain high stability under convenient storage conditions (4 degrees Celsius), enhancing its usability in lab conditions.

Conclusion

• The researchers conclude that the constructed CEM-POS recombinant plasmid can serve as a valuable tool for improving the accuracy and robustness in routine CEM-PCR examinations because it serves as a reliable reaction control and mitigates the risks of false positives.