CMP^(TM) MycoBeads抗體磁珠套組應用於分枝桿菌的培養與real-time PCR檢測效能

Abstract

The detection performance of acid-fast stain could be promoted by using CMP^(TM) MycoBeads to concentrate mycobacteria in sputum specimens. The concentrated sputum specimens are further applied to the performance assessment of the culture and real-time PCR tests. In the culture test, clinical isolated Mycobacterium tuberculosis complex (MTBC), which includes 20 Mycobacterium tuberculosis and 10 Mycobacterium bovis stains were mixed individually in quantities of 1 x 10^5 CFU with high microbial sputum to simulate highly contaminated clinical specimens. The specimens were pretreated by usual clinical practice to compare the concentration efficacy of mycobacteria with centrifugation method and the CMP^(TM) MycoBeads method. After the sputum specimens were digested to liquid form, mycobacteria were concentrated from sputum using both methods, and then inoculated on L-J medium slant and Middlebrook 7H9 broth. After incubation in suitable conditions, i.e. until the colonies appeared, the growth status for contamination and mycobacteria detection rates were evaluated. Furthermore, CMP^(TM) MycoBeads were used to concentrate six other non-tuberculosis mycobacteria (NTM) and, then, inoculated on the L-J medium slant. The results indicated that CMP^(TM) MycoBeads effectively reduced the contamination rate and increased the MTBC and NTM detection rates. Results also indicated that CMP^(TM) MycoBeads bind to all NTM strains tested, but the binding efficiency is slightly worse than for the MTBC. In the real-time PCR method for mycobacteria detection, after specimens were digested by neutral digestion solution (NALCsodium citrate solution without 2.5% NaOH) and concentrated by both methods, the MTBC DNA were extracted and detected in the real-time PCR test. The differences in Ct value (the smaller value represent the replicate signal reach threshold earlier) and △Rn (the intensity of fluorescence; the higher value of △Rn the easier for interpretation) from each concentration method were compared. The results showed that, in comparison with the centrifugation method, the CMP^(TM) MycoBead method lowered the Ct value by 3.8~4.1% and enhanced △Rn to 49.7~52.3%. The CMP^(TM) MycoBead and centrifugation methods were also used with two biochip Identification kits, GenoType® Mycobacterium CM (Hain Lifescience GmbH, Germany) and CMP^(TM) MycoChip (Creative Microbiologicals, Ltd., Taiwan). The imaging software analysis showed that the CMP^(TM) MycoBead method enhanced signal intensity more than the centrifugation method. Based on this finding, we conclude that the CMP^(TM) MycoBeads can provide an efficient mycobacteria concentration method that reduces culture contamination and increases the sensitivity of mycobacteria detection by either real-time PCR method or the biochip method for those TB laboratories without a centrifuge facility or have not yet integrated the centrifugation method into their routine practice.