Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction.
Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction.
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DOI：
10.1186/cc702
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Vanderkelen A
中科院分区：
文献类型：
--
作者：
Pirnay JP;De Vos D;Duinslaeger L;Reper P;Vandenvelde C;Cornelis P;Vanderkelen A
我们开发了一种具有快速热循环功能的实时检测 (RTD) 聚合酶链反应 (PCR)，用于检测和定量伤口活检样本中的铜绿假单胞菌。该方法产生了 7 个对数的线性定量检测范围，检测下限为 103 个菌落形成单位 (CFU)/g 组织或每次反应几个拷贝。从样本采集到出结果的时间不到1小时。 RTD-PCR 具有快速定量检测重症监护患者病原体的潜力，从而实现早期和个体化治疗。伤口定植的早期诊断或伤口脓毒症的预测为治疗干预提供了机会。需要比细菌培养更快的定性和定量测试。铜绿假单胞菌导致高发病率和死亡率，对常见抗生素具有固有耐药性，并且越来越多地作为医院病原体被分离出来。我们开发了三种基于 PCR 的方法来检测和定量伤口活检样本中的铜绿假单胞菌：常规 PCR、酶联免疫吸附测定 (ELISA)-PCR 和快速热循环 RTD-PCR（LightCycler™ 技术），所有方法均基于外膜脂蛋白基因 oprL 的扩增。我们通过定量测量连续稀释液、重构皮肤样本和 21 个烧伤创面活检样本中的铜绿假单胞菌水平，将这些方法与细菌培养的功效进行比较。从过夜的铜绿假单胞菌培养物中制备连续 10 倍稀释液，并将其铺在 Luria-Bertani 和西曲溴铵琼脂平板上。将琼脂板在 37°C 下孵育过夜，并对菌落进行计数，以估计每个稀释管的 CFU 数量。从每个稀释管中取出样品作为三种基于 PCR 的方法的模板。将系列铜绿假单胞菌稀释液（见上文）添加到未感染的尸体皮肤中。使用组织撕裂器均质化重构的活检样本，并使用 XTRAX DNA 缓冲液提取 DNA。将DNA重悬于蒸馏水中。样本被用作基于 PCR 的方法的模板。从 9 名疑似铜绿假单胞菌烧伤创面感染的患者身上采集了 21 份烧伤创面活检样本。将活检样本纵向分成两块。从一块中提取 DNA（使用 XTRAX DNA 缓冲液）并用作基于 PCR 的技术的模板（见上文）。使用组织撕裂器将另一块在生理水中均质化。将悬浮液的连续 10 倍稀释液涂在 Luria-Bertani 和西曲溴铵琼脂平板上。 37°C 孵育过夜后进行菌落计数。 PCR 混合物含有无菌蒸馏水、PCR 缓冲液、脱氧核苷酸混合物或地高辛标记混合物、MgCl2、稀释模板、引物 PAL1 和 PAL2 以及 AmpliTaQ DNA 聚合酶。扩增在 GeneAmp® PCR System 2400 中进行。将反应混合物的等分试样置于琼脂糖凝胶上进行电泳和 PCR 产物的可视化。使用数码相机拍摄凝胶图像。使用图像分析软件计算实验条带的条带质量。将地高辛标记反应的等分试样变性，然后与生物素化捕获探针 PrL 杂交。将部分所得溶液转移至链霉亲和素包被的微量滴定板 (MTP) 的孔中，并在 45°C 下孵育 3 小时。丢弃该溶液。添加过氧化物酶缀合的抗地高辛，并将 MTP 在 37°C 下孵育 30 分钟。弃去溶液并添加ABTS底物。 MTP 在 37°C 下孵育 30 分钟。在 405 nm 处读取吸光度。 RTD-PCR 混合物含有 PCR 级无菌水、稀释的模板 DNA、引物 PAL1 和 PAL2、3' 荧光素 (FL) 标记探针 oprL-FL、5' LC 红色 640 标记和 3' 磷酸化探针 oprL-LC、MgCl2 和 LC DNA Master 杂交探针，含有 Taq DNA 聚合酶、反应缓冲液、dNTP 与 dUTP 混合，而不是dTTP 和 MgCl2。扩增在 LightCycler™ 中进行。在退火阶段测量 LC Red 640 的荧光信号。使用分析软件对测量的荧光数据进行处理。三种方法与培养结果具有良好的一致性。传统 PCR 的灵敏度至少比细菌培养低 100 倍，并且动态范围较低（2 个对数）。 ELISA-PCR 的检测下限为 103 CFU/g 组织，其灵敏度是传统 PCR 的十倍。然而，动态范围并没有增加。 ELISA-PCR 非常耗时（8 小时）。 RTD-PCR 产生的线性定量检测范围为 7 个对数，检测下限为 103 CFU/g 组织。但更重要的是，从样本采集到得出结果的时间不到1小时。两个活检标本在 ELISA-PCR 和 RTD-PCR 中的得分显着高于细菌培养中的得分。这可能表明死亡细菌的 DNA 被扩增。所有基于 PCR 的方法均发现十分之一的培养阳性活检样本呈阴性。局部抗菌剂可能会抑制 PCR。这些结果表明，RTD-PCR 具有快速定量检测重症监护患者病原体的潜力，从而实现早期和个体化治疗。需要进一步研究来评估这项新技术的可靠性及其对患者治疗结果和医院成本的影响。
We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Early diagnosis of wound colonisation or prediction of wound sepsis provides an opportunity for therapeutic intervention. There is need for qualitative and quantitative tests that are more rapid than bacterial culture. Pseudomonas aeruginosa results in high morbidity and mortality rates, is inherently resistant to common antibiotics, and is increasingly being isolated as a nosocomial pathogen. We developed three PCR-based methods to detect and quantify P aeruginosa in wound biopsy samples: conventional PCR, enzyme-linked immunosorbent assay (ELISA)-PCR, and RTD-PCR with rapid thermal cycling (LightCycler™ technology), all based on the amplification of the outer membrane lipoprotein gene oprL. We compared the efficacy of these methods to bacterial culture by quantitatively measuring levels of P aeruginosa in serial dilutions, in reconstituted skin samples and 21 burn wound biopsy samples. Serial 10-fold dilutions were made from an overnight P aeruginosa culture and plated out onto Luria-Bertani and cetrimide agar plates. The agar plates were incubated overnight at 37°C, and the colonies were counted in order to estimate the number of CFU per dilution tube. A sample was taken from each dilution tube as a template for the three PCR-based methods. Serial P aeruginosa dilutions (see above) were added to uninfected cadaveric skin. The reconstituted biopsy samples were homogenized using a tissue tearer and DNA was extracted using XTRAX DNA buffer. The DNA was resuspended in distilled water. A sample was taken as a template for the PCR-based methods. Twenty-one burn wound biopsy samples were taken from nine patients with suspected P aeruginosa burn wound infection. The biopsy samples were longitudinally divided into two pieces. From one piece, DNA was extracted (using XTRAX DNA buffer) and used as a template for PCR-based techniques (see above). The other piece was homogenized, in physiological water, using a tissue tearer. Serial 10-fold dilutions of the suspension were spread on Luria-Bertani and cetrimide agar plates. Colony counts were performed after overnight incubation at 37°C. The PCR mixture contained sterile distilled water, PCR buffer, deoxynucleotide mixture or digoxigenin labelling mix, MgCl2, diluted template, primers PAL1 and PAL2, and AmpliTaQ DNA polymerase. The amplification was performed in a GeneAmp® PCR System 2400. An aliquot of the reaction mixture was put on an agarose gel for electrophoresis and visualisation of the PCR product. An image of the gel was made using a digital camera. Image analysis software was used to calculate the band mass of the experimental bands. An aliquot of the digoxigenin labelling reaction was denatured and then hybridized with the biotinylated capture probe PrL. Some of the resultant solution was transferred to a well of a streptavidin-coated microtitre plate (MTP) and incubated for 3 h at 45°C. The solution was discarded. Peroxidase conjugated antidigoxigenin was added and the MTP was incubated for 30 min at 37°C. The solution was discarded and ABTS substrate was added. The MTP was incubated for 30 min at 37°C. Absorbance was read at 405 nm. The RTD-PCR mixture contained PCR grade sterile water, diluted template DNA, primers PAL1 and PAL2, 3' fluorescein (FL)-labelled probe oprL-FL, 5' LC Red 640-labelled and 3' phosphorylated probe oprL-LC, MgCl2, and LC DNA Master Hybridisation Probes, containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, and MgCl2. The amplification was performed in a LightCycler™. The fluorescence signal of LC Red 640 was measured during the annealing phase. The measured fluorescence data was processed with analysis software. The three methods showed a good concordance with the culture results. Conventional PCR was at least 100 times less sensitive than bacterial culture and had a low dynamic range (2 logs). With a lower detection limit of 103 CFU/g tissue, ELISA-PCR was ten times more sensitive than conventional PCR. The dynamic range, however, did not increase. ELISA-PCR is very time consuming (8 h). The RTD-PCR produced a linear quantitative detection range of 7 logs with a lower detection limit of 103 CFU/g tissue. More important, however, was that the time from sample collection to result was less than 1 h. Two biopsy specimens scored significantly higher in ELISA-PCR and RTD-PCR than in bacterial culture. This could indicate that DNA from dead bacteria was amplified. One out of ten culture positive biopsy samples was found negative by all PCR-based methods. Topical antimicrobial agents possibly inhibited PCR. These results show that RTD-PCR has potential for the rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment. Further study is required to assess the reliability of this new technology, and its impact on patient outcome and hospital costs.
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DOI：
10.1073/pnas.88.16.7276
发表时间：
1991-08-01
影响因子：
11.1
作者：
HOLLAND, PM;ABRAMSON, RD;GELFAND, DH
通讯作者：
GELFAND, DH
影响因子：
2.8
作者：
Lim, A;DeVos, D;Cornelis, P
通讯作者：
Cornelis, P
影响因子：
9.4
作者：
DOERN, GV;VAUTOUR, R;LEVY, B
通讯作者：
LEVY, B
影响因子：
5.4
作者：
Lewin, SR;Vesanen, M;Markowitz, M
通讯作者：
Markowitz, M
影响因子：
9.4
作者：
Martell, M;Gómez, J;Guardia, J
通讯作者：
Guardia, J