以核酸染劑結合聚合酶連鎖反應定量空氣中活性細菌

Abstract

空氣中存在著許多對人體健康有害的物質，懸浮於空氣中的微生物 (生物氣膠 (bioaerosols))即是其中之一。近年來許多針對室內及室外作業場所生物氣膠之研究，大部分多使用培養法作為其分析環境中總細菌濃度之方法；然而已知生物氣膠具有活性但不可培養 (viable but non-culturable (VBNC))之特性，因此造成以培養法分析時低估實際暴露濃度。 本研究透過核酸染劑結合即時定量聚合酶連鎖反應 (real-time quantitative polymerase chain reaction, qPCR)，建立定量空氣中活性細菌的方法。並比較不同的核酸染劑(ethidium monoazide (EMA) 及propidium monoazide (PMA))以及不同的濃度之效能、測試其線性關係範圍，並於高濃度細菌暴露之職場進行方法驗證。 於核酸染劑結合qPCR結果顯示，相較於未添加核酸染劑之對照組，加入EMA或PMA之受熱組樣本，其qPCR所得之DNA濃度顯著較低，顯示EMA與PMA皆可有效抑制受熱組細菌DNA於qPCR之放大。而於未受熱組之樣本，無EMA處理者其DNA量為6.09 log copies/μL，有EMA處理者為2.78~4.58 log copies/μL，顯示EMA會干擾活性菌定量；而另一核酸染劑PMA於未受熱組之結果，無PMA處理之DNA量為5.99 log copies/μL，而有PMA處理者為4.41~5.41 log copies/μL，其差異介於0.57~1.57 log copies/μL，顯示PMA較不會影響活性細菌之DNA於qPCR監測。而進一步觀察不同濃度核酸染劑間之反應，結果顯示濃度為1.5 μg/mL之PMA可有效抑制受熱組細菌DNA於qPCR放大，且在此濃度下，不會對活性細菌造成以qPCR定量細菌濃度之明顯干擾。因此以1.5 μg/mL PMA結合qPCR作為定量空氣中活性細菌之最佳條件。 進一步以最佳核酸染劑條件1.5 μg/mL PMA評估偵測細菌濃度範圍，結果顯示其線性範圍介於1×104 ~ 1×1010 cfu/mL (R2=0.9945)。另考慮每種細菌之copy number不同，無法直接將qPCR所得之copies數換算細菌數，因此本研究亦將四種環境地點(醫療院所、木材行、豬舍及雞舍)之樣本以qPCR分析(copies)及Baclight計數(cells)建立可適用於總細菌與活性細菌之環境檢量線，在總細菌之檢量線為Y (log cells/sample)=1.034 X (log copies/sample)-0.6278 (R2=0.9592)；活性細菌之檢量線為Y (log cells/sample)=1.085 X (log copies/sample)-0.8618 (R2=0.9665)，高R2顯示此兩條檢量線可做為細菌數之換算依據。最後將開發之方法應用於分析職業場所空氣採樣之樣本，其量測場所包括醫療院所、木材行、水稻田、蔬菜田、家禽舍及畜牧業，而將各地點所測得之細菌濃度以Wilcoxon signed-rank進行檢定，結果顯示五個地點之總細菌濃度皆大於活性細菌濃度(P<0.0001)，活性細菌濃度亦大於可培養細菌濃度(P<0.0001)，因此本研究認為以1.5 μg/mL PMA結合qPCR作為定量職業環境空氣中活性細菌是可行的分析方法。

Microbial contamination in air has gained particular attention primarily due to the adverse human health effects associated with bioaerosols. The recent studies on monitoring the bioaerosols in occupational settings are commonly based on the analysis with culture assay. However, it has been known that airborne bacteria may be present as viable but not culturable (VBNC) state; therefore, the quantification of airborne bacteria by culture assay may underestimate the actual exposure level. To deal with this problem that both culture assay and qPCR may not accurately quantify the level of total viable bacteria, this study is initiated to develop a qPCR-based method coupled with nucleic acid dye (ethidium monoazide and propidium monoazide) that can exclusively quantify total viable bacterial. Different types and concentrations of nucleic acid dyes were evaluated. The most appropriate selection on nucleic acid dye was tested for the range of the detection limit, and furthermore, it was pretested in several occupational environments with the high level of total bacteria exposure. The results of qPCR-based method coupled with nucleic acid dye, for dead cells, a significant decrease of DNA concentration was observed for the heated cells pretreated with EMA or PMA as compared to untreated samples. Indicated that EMA and PMA can penetrate dead bacteria and inhibited the DNA amplification in qPCR. For live cells, untreated samples DNA concentration were 6.09 log copies/ul. EMA-treated samples were 2.78~4.58 log copies/ul, indicated that EMA can penetrated viable bacteria and decrease the viable cells concentration. For PMA, untreated samples DNA concentration were 5.99 log copies/ul. Otherwise, PMA-treated samples were 0.57~1.57 log copies/ul, there was showed no statistical significant difference in DNA concentration between PMA-treated and -untreated samples. These results demonstrated that PMA penetrated dead bacteria and inhibited the DNA amplification in qPCR, indicating that PMA coupled with qPCR is applicable to quantify the live bacteria. Moreover, the DNA concentrations measured in dead cells were independent of PMA at a concentration of 1.5 μg/mL. Thus, PMA at 1.5 μg/ml with a 20-min light exposure was considered as the most suitable for quantification and thus recommended for all the following experiments in this study. As for testing on the detection limit of the PMA-qPCR assay, a linear range was obtained for the cells from 1×104 to 1×1010 cfu/mL (R2=0.9945). To conceder bacteria have different copy number, Can’t direct use the copies number conversion bacterial number. Therefore, this study also sample four environmental (), for qPCR analysis (copies) and Baclight count (cells) to establish the applicable total bacteria and viable bacteria in environmental calibration curve. Total bacteria calibration curve was Y (log cells/sample)=1.034 X(log copies/sample)-0.6278 (R2=0.9592); for viable bacteria calibration curve was Y (log cells/sample)=1.085 X (log copies/sample)-0.8618 (R2=0.9665). Using the calibration curve to translation from copies number to cell numbers. Finally, this PMA-qPCR based methodology was used for analyzing the samples collected from the air of the workplaces including the hospital, sawmill, rice paddy fields, vegetable fields, poultry and swine, Various workplaces samples measured concentration of bacteria use the Wilcoxon signed-rank test, the results show that the concentration of total bacteria of the five sites are greater than the viable bacteria concentration (p<0.0001), viable bacteria concentration are greater than the concentration of culturable bacteria (p<0.0001). Therefore PMA-PCR assay was revealed as suitable for quantification of viable bacteria in those environments.