Collins S, Jorgensen F, Willis C, Walker J
Public Health England, Biosafety Investigation Unit, Salisbury, UK.
Public Health England, Food, Water and Environmental Microbiology Laboratory, Salisbury, UK.
J Appl Microbiol. 2015 Oct;119(4):1158-69. doi: 10.1111/jam.12911. Epub 2015 Sep 4.
Culture remains the gold-standard for the enumeration of environmental Legionella. However, it has several drawbacks including long incubation and poor sensitivity, causing delays in response times to outbreaks of Legionnaires' disease. This study aimed to validate real-time PCR assays to quantify Legionella species (ssrA gene), Legionella pneumophila (mip gene) and Leg. pneumophila serogroup-1 (wzm gene) to support culture-based detection in a frontline public health laboratory.
Each qPCR assay had 100% specificity, excellent sensitivity (5 GU/reaction) and reproducibility. Comparison of the assays to culture-based enumeration of Legionella from 200 environmental samples showed that they had a negative predictive value of 100%. Thirty eight samples were positive for Legionella species by culture and qPCR. One hundred samples were negative by both methods, whereas 62 samples were negative by culture but positive by qPCR. The average log10 increase between culture and qPCR for Legionella spp. and Leg. pneumophila was 0·72 (P = 0·0002) and 0·51 (P = 0·006), respectively.
The qPCR assays can be conducted on the same 1 l water sample as culture thus can be used as a supplementary technique to screen out negative samples and allow more rapid indication of positive samples.
The assay could prove informative in public health investigations to identify or rule out sources of Legionella as well as to specifically identify Leg. pneumophila serogroup 1 in a timely manner not possible with culture.
培养法仍是环境中军团菌计数的金标准。然而，它存在一些缺点，包括孵育时间长和灵敏度低，导致对军团病暴发的响应时间延迟。本研究旨在验证实时荧光定量聚合酶链反应（qPCR）检测方法，以定量军团菌属（ssrA基因）、嗜肺军团菌（mip基因）和嗜肺军团菌血清1型（wzm基因），以支持一线公共卫生实验室基于培养的检测。
每种qPCR检测方法均具有100%的特异性、出色的灵敏度（5基因组单位/反应）和可重复性。将这些检测方法与基于培养的200份环境样本中军团菌计数进行比较，结果显示它们的阴性预测值为100%。38份样本通过培养和qPCR检测，军团菌属呈阳性。两种方法检测均为阴性的样本有100份，而62份样本培养检测为阴性，但qPCR检测为阳性。军团菌属和嗜肺军团菌培养与qPCR之间的平均log10增加值分别为0.72（P = 0.0002）和0.51（P = 0.006）。
qPCR检测可与培养在同一1升水样上进行，因此可作为一种补充技术，筛选出阴性样本，并更快地指示阳性样本。
该检测方法在公共卫生调查中可能具有重要意义，有助于识别或排除军团菌来源，并能及时特异性地鉴定嗜肺军团菌血清1型，这是培养法无法做到的。
