A Meta-Analysis Of Ureaplasmas Infection And Nongonococcal Urethritis And Premilinary Establishment Of Detecting Ureaplasmas With PCR-ELISA

ObjectiveInvestigate the correlation between Ureaplasma urealyticum, Ureaplasma parvum infection and male nongonococcal urethritis. By the retrospective study, explore the Uu, Up different virulence in the male nongonococcal urethritis, which will provide the important reason for developing a method, which can detect and differentiate the Ureaplasmas at the same time. Accoeding to what is said above, we attempt to combines the PCR, nucleic acid hybridization and enzyme color technology and establish a technology named PCR-ELISA to detect and differentiate the Ureaplasma urealyticum, Ureaplasma parvum.MethodsThe studies on the relationship of Ureaplasmas infection and male nongonococcal urethritis were collected by searching from Pubmed database,CBM database,CNKI database and so on(2000-2011), and analyzed by meta-analysis. The odds ratio was calculated by Review Manager5.0software. In the second section, we use the biotin-labelled primer pair (U4, U5) amplified the Ureaplasma urealyticum and Ureaplasma parvum by polymerase chain reaction (PCR). Search for the PCR product’s nucleic acid sequence by the Primer online program and designed the special capture probes to Ureaplasma urealyticum and Ureaplasma parvum. Bind the capture probes to the DNA-BINDTM microplate. The biotin-labelled PCR denatured products were hybridized with the capture probes. Then the biotin reacted with the HRP-Streptavidin system and a colorimetric assay was performed. Last, integrated the procedures and established the PCR-ELISA method. Meanwhile, the specificity testing was determined by using the reference bacterias such as Mycoplasma genitalium G37, Beta hemolytic streptococcus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus and Up standerd strain serotype3, Uu standard strain serotype8. In addition, the38clinical swabs were randomly selected from the Second Hospital of Tianjin Medical University to assess the method’s clinical feasibility.ResultsEleven studies accorded with research criteria including1824cases and1584 controls were analyzed. The combined OR value of Ureaplasmas infection to male NGU is1.23,95%CI(0.91,1.68), the test for overall effect P=0.18.However, the OR value of Uu infection to male NGU is1.65,95%CI(1.10,2.47), the test for overall effect P=0.02. But, contrary to Uu, the OR value of Up infection to NGU is0.57,95%CI(0.45,0.72), the test for overall effect P<0.00001. In the second section, the capture probes to differentiate the Ureaplasma urealyticum and Ureaplasma parvum were designed as followed:Uu capture probe:5’AGC AAT TAA CTT CGC TGA AGG C3’; Up capture probe:5’GGC AAT TAA TTT CGC TAG TGG T3’. Soon afterwards, the PCR-ELISA method was preliminarily established by applying the U4, U5primer pair, the capture probe and the DNA-BINDTM microplate. The method was specific so that only the Uu and Up can be detected and discriminated in the specificity testing. Meanwhile in the clinical feasibility testing, the PCR-ELISA method succeeded to detect and differentiate the38clinical swabs. Among the38clinical swabs, the positive specimens were26, comprising of the Up infections21, Uu infections3, Up, Uu co-infections2and negative specimens12. The positive rate was68.42%. Compared to conventional culture, the Kappa value was0.687. There was a certain extent consistency between the two methods. Meanwhile, using culture method as a standard, the PCR-ELISA method’sensitivity is88.89%(24/27), the specificity81.82%(9/11).ConclusionsUu mainly exists in the NGU group. Contrast to Up, Uu has higher virulence and is the one of male NGU virulent agents. However, Up has the lower virulence and is the parasitic microorganism in the male urethra. So, it is important to detect separately Ureaplasma urealyticum and Ureaplasma parvum. It will have far-reaching influence for epidemiological survey and mechanism study. The PCR-ELISA assay is a technology, which combines the PCR, nucleic acid hybridization and enzyme color technology. The PCR-ELISA can not only effectively dectet the Ureaplasmas like the culture method, but also differentiate the Uu and Up. The procedure is simple, the reagent is easily obtained from the commercial products and no special machine is needed. In this study, only a pair of capture probe is used to discriminate the pathogenic microorganism. Compared to previous PCR-ELISA, the digoxin and anti- digoxin antibody are not needed, so this not only reduce the procedure but also lower the cost. The PCR-ELISA method is suit to be applied to the secondary hospital and simple microorganism laboratory. The PCR-ELISA method is complemental to culture method and it will provide a new approach for the detecting and differentiating of Ureaplasma urealyticum and Ureaplasma parvum.