Rapid microbial sample preparation from blood using a novel concentration device.
Rapid microbial sample preparation from blood using a novel concentration device.
复制标题
DOI：
10.1371/journal.pone.0116837
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Sauer-Budge AF
中科院分区：
文献类型：
--
作者：
Boardman AK;Campbell J;Wirz H;Sharon A;Sauer-Budge AF
对菌血症患者的适当护理取决于做出准确诊断所需的时间。然而，这些患者血液中的微生物浓度极低(1-100cfu/ml)，传统上需要培养(血培养)或扩增(例如，聚合酶链式反应)进行检测。目前基于培养的方法可能至少需要两天时间，而更快的方法，如聚合酶链式反应(PCR)，需要的样本不含抑制物(即血液成分)。尽管存在从这些样本中提取血液的商业试剂盒，但它们通常只捕获DNA，因此有必要使用血液培养进行抗菌素测试。在这里，我们报告了一种新的、放大的样品制备方法，该方法是在一种新的微生物浓缩装置上进行的。该工艺可以有效地溶解10毫升细菌血，同时保持微生物的生存能力，从而获得30-μL的最终输出量。一套六种微生物(金黄色葡萄球菌、肺炎链球菌、大肠杆菌、流感嗜血杆菌、铜绿假单胞菌和白色念珠菌)在一系列临床相关浓度下进行了测试。在最高检测浓度为100cfu/m L时，所有微生物的回收率均大于55%，其中3种微生物的回收率超过70%。在最低检测浓度为3cfu/m L时，两种微生物的回收率约为40-50%，而其他四种微生物的回收率均大于70%。使用对甲氧西林敏感的金黄色葡萄球菌(MSSA)的Taqman试验来证明下游分析的可行性，我们证明了我们的微生物颗粒足够干净，可以进行PCR扩增。对56份加标阳性和阴性样本的PCR检测，其特异性为0.97，敏感性为0.96，表明我们的样本制备方法在直接从原始样本快速诊断菌血症方面具有很大的前景。
Appropriate care for bacteremic patients is dictated by the amount of time needed for an accurate diagnosis. However, the concentration of microbes in the blood is extremely low in these patients (1–100 CFU/mL), traditionally requiring growth (blood culture) or amplification (e.g., PCR) for detection. Current culture-based methods can take a minimum of two days, while faster methods like PCR require a sample free of inhibitors (i.e., blood components). Though commercial kits exist for the removal of blood from these samples, they typically capture only DNA, thereby necessitating the use of blood culture for antimicrobial testing. Here, we report a novel, scaled-up sample preparation protocol carried out in a new microbial concentration device. The process can efficiently lyse 10 mL of bacteremic blood while maintaining the microorganisms’ viability, giving a 30‑μL final output volume. A suite of six microorganisms (Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, and Candida albicans) at a range of clinically relevant concentrations was tested. All of the microorganisms had recoveries greater than 55% at the highest tested concentration of 100 CFU/mL, with three of them having over 70% recovery. At the lowest tested concentration of 3 CFU/mL, two microorganisms had recoveries of ca. 40–50% while the other four gave recoveries greater than 70%. Using a Taqman assay for methicillin-sensitive S. aureus (MSSA)to prove the feasibility of downstream analysis, we show that our microbial pellets are clean enough for PCR amplification. PCR testing of 56 spiked-positive and negative samples gave a specificity of 0.97 and a sensitivity of 0.96, showing that our sample preparation protocol holds great promise for the rapid diagnosis of bacteremia directly from a primary sample.
登录
查看更多内容
影响因子：
4.6
作者：
I-Fang Cheng;Chang, Hsien-Chang;Chen, Tzu-Ying;Hu, Chenming;Yang, Fu-Liang
通讯作者：
Yang, Fu-Liang
影响因子：
8.8
作者：
Gaieski, David F.;Mikkelsen, Mark E.;Goyal, Munish
通讯作者：
Goyal, Munish
影响因子：
9.4
作者：
HERLICH, MB;SCHELL, RF;LEFROCK, JL
通讯作者：
LEFROCK, JL
影响因子：
8.8
作者：
Kumar, Arland;Roberts, Daniel;Cheang, Mary
通讯作者：
Cheang, Mary
DOI：
10.1073/pnas.77.8.4914
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
HOFFMAN, RA;KUNG, PC;GOLDSTEIN, G
通讯作者：
GOLDSTEIN, G