[Research on ZNA probe in the quantitative detection of chlamydia trachomatis nucleic acid]

Objective: To investigate the performance of ZNA(ZIP Nucleic Acid) probes and its application in the quantitative detection of Chlamydia trachomatis (CT)nucleic acid. Methods: Use CT positive plasmids to compare the PCR amplification curves of ZNA probes coupled with different ZIP numbers. Compare ZNA probes with other three sets of probes [namely, 29mer ordinary Taqman probes (long-DNA probe), 20mer ordinary Taqman probes (short-DNA probe) and MGB probes] for stability in PCR amplification curves and repeated freezing and thawing, and the difference in the detection rate of low-concentration plasmids. Use CT positive clinical samples to compare the difference in amplification curves between ZNA probes, long-DNA probe, short-DNA probe and MGB probes, and the detection rate of low-concentration samples. Results: (1) The Ct value and fluorescence value of the probes coupled with 5ZIP units are both better than those coupled with a smaller number of ZIPs. And the difference is biggest when compared with only coupled with 1 ZIP unit: Ct value increased by 1.34 (sensitivity increased by 2.37 times), and fluorescence value increased by 30%. (2) The amplification efficiency of the ZNA probe coupled with 5 ZIPs is 2.14-2.64 times that of the preferred ordinary Taqman probe and MGB probe, and the fluorescence value is 17%-90% higher. (3) The probe freeze-thaw stability results show that the ZNA probe has the best stability, and the lowest concentration of Ct value has the smallest deviation (CV% = 1.4), which is better than the other three sets of probes (CV%=1.7-3.7). (4) Using 35 CT positive clinical samples to compare the PCR amplification performance, compared with other three sets of probes, the amplification sensitivity of ZNA probes was increased by 1.60, 0.99 and 1.06 times respectively. And the results of the consistency analysis of the Ct value show that compared with short-DNA probe and MGB probes, ZNA probes have better detection performance for clinical samples. (5) Use low concentration plasmid template (200, 100, 50 and 10 copies/mL respectively) to compare the amplification sensitivity of the four sets of probes, the detection rate of ZNA probe is the best. Especially, at the lowest concentration 10 copies/mL, the detection rate of the other sets of probes is only 15%-20%, but the ZNA probe is still 30%. (6) In 20 clinical samples with different low concentrations (200, 150, 100, and 50 copies/mL), the detection rate of ZNA probes was the highest, which were 100%, 95%, 90%, and 70%, respectively. Conclusions: Through testing of the amplification efficiency, fluorescence value, freeze-thaw stability, the amplification performance of clinical samples and the detection sensitivity of low-concentration samples, ZNA probes coupled with 5 ZIPs show better performance than ordinary Taqman probes and MGB probes. As a new probe technology with flexible design and easy synthesis, ZNA probe can further improve detection sensitivity of low concentration samples in the field of gene expression.