Establishment Of Phage Amplified Biologically Assay By Using Mycobacteriophage Chy2and Evaluation Of Its Capacity Of Detecting Of Mycobacterium Tuberculosis In Sputum Samples

ObjectiveTuberculosis is a severe human-threatening public infectious diseasewhich is characterized by high prevalence, high morbidity and highmortality worldwide. Early detection, rapid diagnosis and effective, timelymedications are important to cut down the chances of communication andbring the spread of the epidemic situation under control. Phage amplifiedbiologically assay（PhaB） is a new-phenotype etiological testing technologycapable of fast detection of live M. tuberculosis by usingmycobacteriophage D29. Low cost, easy operation, high specificity andreporting results within48hours endow the PhaB with vast developmentalspace and significant potential applicaton, especially beneficial for thehigh-load tuberculosis but under-developmental countries and regions.However, the main weakness of PhaB exists in its low sensitivity, whichinhibits a wide-region application of the technology.Through our pilot study of the Major Project of the “Eleventh Five-year Plan” of National Science and Technology, our laboratory isolated andidentified a new mycobacteriophage named Chy2for the first timenationwide. Based on the research outcome, combined with the geneticbackground, biological characteristics of Chy2and on account of the keylinks of PhaB, this study aims to establish PhaB by using Chy2insubstitution for D29and discuss its detective efficacy of M.tuberculosis insputum samples, laying the foundation of utilization of Phage Chy2andimprovement of PhaB.Methods1. M.smegmatis was cultured by liquid culture medium. M. tubercu-losisstandard strain was cultured by L-J culture medium. Both concentrationwas measured by colony-counting method. Double-plating culture methodwas used to amplify phage. The titer of phage was measured by small platecounting method.2. The lowest acting concentration and shortest acting time of phage-inactivation agents were tested by spot tests, to confirm the most effectiveacting condition of phage-inactivation agents to phage Chy2.3. The optimal infectious time and titer of phage to MTB and MSwere confirmed by plague counts respectively.4.58clinical sputum samples were collected and pretreated. PotentialM.tuberculosis in the samples was tested by Chy2-PhaB and D29-PhaBrespectively, with modified L wenstein-Jensen culture method as the diagnostic standard. The detective accuracy of the both assays wascomparatively analyzed.Results1. The concentration of MS and MTB was4.5×10⁹CFU/ml,3×10⁸CFU/ml，respectively. The titer of Chy2and D29was2.2×10¹¹PFU/ml,1.9×10¹¹PFU/ml, respectively.2. At least6%Ferrous ammonium sulfate solution mixed with Chy2for at least5min was able to completely inactivate the extracellular freephage.3.2.2×10¹¹CFU/mL Chy2incubating with MTB for240min couldinfect MTB most effectively.4. PhaB assay using Chy2to detect MTB in sputum samples onlyneeded48hours. The sensitivity, specificity, Youden index, positivepredictive value and negative predictive value were94.7%、85.0%、0.797、0.923、0.895, respectively, while the equivalent corresponding results forD29PhaB assay were76.3%、95.0%、0.713、0.967、0.678, respectively.Conclusion1. The key process of PhaB assay in substitution Chy2for D29wasexplored, thus leading to the successful establishment of the Chy2-PhaB.2. Chy2-PhaB has a higher sensitivity and NPV compared to D29-PhaB.3. The specificity, Youden index and PPV of Chy2-PhaB and D29- PhaB have no significant difference.