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

The Performance Assessment on CMP^(TM)MycoBead to Mycobacteria Culture and Real-Time PCR Method

邱彥昕(Yen-Hsin Chiu) ； 羅晟展(Cheng-Chan Lo) ； 何耿德(Gan-Der Ho) ； 蔡岳廷(Yueh-Ting Tsai) ； 吳金洌(Jen-Leih Wu)

《檢驗及品保雜誌》 1卷3期 (2012/08) Pp. 99-110

https://doi.org/10.6573/JTQA.201208_1(3).01

摘要

利用CMP^(TM) MycoBeads分枝桿菌抗體磁珠套組（啟新生物科技有限公司，台灣）濃縮純化痰檢體中的分枝桿菌後可提升抗酸性染色鏡檢的偵測效能，進一步將濃縮後檢體進行培養與即時核酸擴增（real-time PCR）檢測的效能評估。在培養實驗中分別使用臨床分離MTBC（Mycobacterium tuberculosis complex）包括20株結核分枝桿菌（Mycobacterium tuberculosis）與10株牛型分枝桿菌（Mycobacterium bovis），將所有測試菌皆以1 x 10^5 CFU混合含大量雜菌之痰液模擬高汙染臨床檢體狀態，以比較不同濃縮方法對MTBC之專一分離效果，檢體經一般臨床方法進行前處理後，分別以離心法及CMP^(TM) MycoBeads進行濃縮，再滴種於Lowenstein-Jensen medium（L-J培養基）以及Middlebrook 7H9液體培養基培養，觀察汙染及生長情形。另外，亦以CMP^(TM) MycoBeads對其他6種非結核分枝桿菌（non-tuberculosis Mycobacteria, NTM）進行濃縮純化後移種L-J培養基，結果指出CMP^(TM) MycoBeads應用於濃縮純化痰檢體以及污染的液體培養基，可有效降低培養汙染率，並增加MTBC及NTM檢出率，結果亦發現CMP^(TM) MycoBeads可抓取所有測試的NTM菌種，惟抓取效率略遜於MTBC。在real-time PCR應用實驗中，以中性消化液（NALC-sodium citrate solution，不含2.5% NaOH）液化檢體後，同樣地分別以離心法及CMP^(TM) MycoBeads法進行濃縮，再萃取分枝桿菌DNA後，以real-time PCR對MTBC進行檢測，比較CT值（越小代表訊號越早出現，檢測靈敏度越高）以及△Rn（螢光訊號強度，代表判讀之容易度）之差異性，結果指出，CMP^(TM) MycoBeads法CT值可下降3.8~4.1%，△Rn可增強49.7~52.3%。另外，將CMP^(TM) MycoBeads法與離心法應用於GenoType® Mycobacterium CM（Hain Lifescience GmbH, Germany）以及CMP^(TM) MycoChip（啟新生物科技有限公司，台灣）兩種晶片鑑定套組，經影像軟體分析顯示，使用CMP^(TM) MycoBeads法可使訊號強度提升，對於MTBC之偵測具有明顯之效益。有鑑於CMP^(TM) MycoBeads可顯著增進分枝桿菌培養檢出率並降低汙染率以及增加分生方法（real-time PCR與晶片）偵測之靈敏度，吾等認為對於無離心設備或尚未使用離心法之TB檢驗室，CMP^(TM) MycoBeads法將可提供另一種有效濃縮分枝桿菌及降低培養污染的檢體處理方式。

關鍵字

分枝桿菌 ； 抗體磁珠 ； 濃縮 ； 純化 ； 培養 ； 即時核酸擴增 ； 晶片

並列摘要

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.

並列關鍵字

Mycobacterium ； antibody magnet bead ； concentration ； culture ； realtime PCR ； chip