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Rapid, sensitive and specific isothermal nucleic acid amplification methods of Chlamydia trachomatis (C. trachomatis) and Neisseria gonorrhoeae (N. gonorrhoeae) have been developed based on cross-priming amplification (CPA). The amplicon of CPA can be detected by a disposable amplicon cross-contamination proof device. The whole assay takes 1–1·5 h from amplification to read out. Ten fold serial dilutions of quantified plasmids were used to test the CPA assay sensitivities, with the detection limits of 45 copies per reaction and 65 copies per reaction for C. trachomatis and N. gonorrhoeae respectively. The specificities of CPA assays for C. trachomatis and N. gonorrhoeae were tested by using total DNA extracted from nine other bacterial strains, and no cross-reactivity was detected. Eighty clinical cervical or vaginal swab specimens were tested by both CPA and real-time polymerase chain reaction (PCR) with the consistencies of 98·75% (79/80) and 97·5% (78/80) for C. trachomatis and N. gonorrhoeae respectively. Using real-time PCR as a reference standard, the clinical sensitivity (positive) and specificity (negative) of CPA was found to be 98·15% (53/54) and 100% (26/26) for C. trachomatis, and 93·75% (30/32) and 100% (48/48) for N. gonorrhoeae. Eight swab specimens tested positive for both C. trachomatis and N. gonorrhoeae simultaneously by real-time PCR and CPA assay. This study demonstrated that CPA is an affordable and accessible assay for C. trachomatis and N. gonorrhoeae detection, with high sensitivity and specificity.Rapid and specific detection of the sexually transmitted pathogens Chlamydia trachomatis (C. trachomatis) and Neisseria gonorrhoeae (N. gonorrhoeae) would enable early treatment and management of their spread. Here, for the first time, a user friendly DNA isothermal amplification method named cross-priming amplification (CPA), was used to test C. trachomatis and N. gonorrhoeae with high sensitivity and specificity. The results indicate that CPA has great potential for improving C. trachomatis and N. gonorrhoeae diagnostics which could be particularly advantageous in resource-limited areas.