Background Due to absence of cost effective and rapid diagnostic test syndromic management of Neisseria and Chlamydia was recommended in developing countries. Being nonspecific such a strategy has resulted in over treatment leading to increased drug resistance. It also misses out on asymptomatic patients resulting in increase in disease burden. The study describes the development and evaluation of a low cost duplex PCR method (dPCR) for co-detection of Neisseria and Chlamydia. Using molecular beacons we further provide a method for quantitative and easy detection of the two pathogens.
Methods Endocervical swabs were collected from patients visiting gynaecology department of various hospitals in Delhi. We standardised and evaluated in-house uniplex PCR (uPCR) for diagnosis of Neisseria against Roche Amplicor Micro Well Plate CT/NG kit. Method was modified to co-detect N. gonorrhoeae and C. trachomatis in single test. Further we developed visual assay for detection of Chlamydia and Neisseria using molecular beacon probe.
Results Clinical samples (n = 412) were used to validate in-house uPCR assay for Neisseria. The PPV and NPV were found to be 86.77% and 97.2%. We further modified our uPCR to dPCR for simultaneous detection of Neisseria and Chlamydia. The overall infection rate was found to be 27.8% and 26.3% for Neisseria and Chlamydia respectively while 11.3% of patients were co-infected. The in-house dPCR was found to be 85.7% sensitive and 97% specific. To further enhance the sensitivity and specificity of our test, molecular beacon were designed against the amplicons. Use of molecular beacons also reduced the detection time as amplicons could be directly visualised under dark reader.
Conclusions The in-house dPCR assay is rapid and as sensitive as commercially available tests. Use of molecular beacons provides a highly specific and easy to use detection method, making it a better option for routine diagnosis of genital infection in developing countries.
- duplex PCR
- Molecular Beacon
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