Rapid and sensitive detection of Pseudomonas aeruginosa in chlorinated water and aerosols targeting gyrB gene using real-time PCR.
Rapid and sensitive detection of Pseudomonas aeruginosa in chlorinated water and aerosols targeting gyrB gene using real-time PCR.
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DOI：
10.1111/j.1365-2672.2011.05107.x
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发表时间：
2011-10
影响因子：
4
通讯作者：
Lee J
中科院分区：
文献类型：
--
作者：
Lee CS;Wetzel K;Buckley T;Wozniak D;Lee J
为了快速检测氯化水和气溶胶中的铜绿假单胞菌，建立了基于gyrB基因的实时PCR检测方法。利用最新的611个假单胞菌和748个其他细菌gyrB基因设计了两个新的引物组（pa722F/746MGB/899R和pa722F/746MGB/788R），以获得高特异性。当对包括囊性纤维化患者临床分离株在内的各种菌株进行检测时，它们的特异性显示出100%的准确性。采用含P. aeruginosa的氯化水和气溶胶模拟水传播和空气传播途径（检出限3.3 × 102 CFU·PCR−1 ~ 2.3 × 103 CFU·PCR−1）。饮用水水平（~ 1 mg·L−1）对实时PCR无氯干扰，但高氯水平（12 mg·L−1）对实时PCR有干扰，需要中和。当雾化器中细菌的初始浓度为104 CFU·mL−1时，用明胶过滤器捕获气溶胶中的铜绿假单胞菌（P. aeruginosa），采样时间最小为2 min。以gyrB基因为靶点，结合优化的样品采集方法和样品处理方法，建立了一种高特异性、快速的检测方法（2 ~ 3小时），可用于氯化水和气溶胶中铜绿假单胞菌的检测，从而在达到所需灵敏度的同时，可以直接从水或气溶胶中提取DNA。新的检测方法可以提供及时和准确的风险评估，以防止铜绿假单胞菌从水和气溶胶暴露，从而减少疾病负担，特别是在免疫功能低下和易感人群中。这种方法可以很容易地用作检测其他类型微生物的平台技术，特别是那些通过水和气溶胶途径传播的微生物，如嗜肺军团菌。
For the rapid detection of P. aeruginosa from chlorinated water and aerosols, gyrB gene-based real-time PCR assay was developed and investigated. Two novel primer sets (pa722F/746MGB/899R and pa722F/746MGB/788R) were designed using the most updated 611 Pseudomonas and 748 other bacterial gyrB genes for achieving high specificity. Their specificity showed 100% accuracy when tested with various strains including clinical isolates from cystic fibrosis patients. The assay was tested with P. aeruginosa-containing chlorinated water and aerosols to simulate the waterborne and airborne transmission routes (detection limit 3.3 × 102 CFU·PCR−1 − 2.3 × 103 CFU·PCR−1). No chlorine interference in real-time PCR was observed at drinking water level (~ 1 mg·L−1), but high level of chorine (12 mg·L−1) interfered the assay, thus neutralization was needed. P. aeruginosa in aerosol was successfully detected after capturing with gelatin filters with minimum 2 min of sampling time when the initial concentration of 104 CFU·mL−1 bacteria existed in the nebulizer. A highly specific and rapid assay (2–3 hrs) was developed by targeting gyrB gene for the detection of P. aeruginosa in chlorinated water and aerosols, combined with optimized sample collection methods and sample processing, so the direct DNA extraction from either water or aerosol was possible while achieving the desired sensitivity of the method. The new assay can provide timely and accurate risk assessment to prevent P. aeruginosa exposure from water and aerosol, resulting in reduced disease burden, especially among immune-compromised and susceptible individuals. This approach can be easily utilized as a platform technology for the detection of other types of microorganisms, especially for those that are transmitted via water and aerosol routes, such as Legionella pneumophila.
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影响因子：
4.2
作者：
Deschaght P;De Baere T;Van Simaey L;Van Daele S;De Baets F;De Vos D;Pirnay JP;Vaneechoutte M
通讯作者：
Vaneechoutte M
影响因子：
5.8
作者：
Larkin, M. A.;Blackshields, G.;Higgins, D. G.
通讯作者：
Higgins, D. G.
DOI：
10.1111/j.1651-2227.1984.tb17774.x
发表时间：
1984-01-01
期刊：
ACTA PAEDIATRICA SCANDINAVICA
影响因子：
--
作者：
GRANSTROM, M;ERICSSON, A;VASIL, ML
通讯作者：
VASIL, ML
影响因子：
--
作者：
ARMSTRONG, DS;GRIMWOOD, K;PHELAN, PD
通讯作者：
PHELAN, PD
影响因子：
3.1
作者：
da Silva, LVF;Tateno, AF;Ramos, SRTS
通讯作者：
Ramos, SRTS