Labrie S, Moineau S
Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie, and Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, Canada G1K 7P4.
Appl Environ Microbiol. 2000 Mar;66(3):987-94. doi: 10.1128/AEM.66.3.987-994.2000.
Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82. 2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10(4) to 10(7) PFU/ml and was 10(3) to 10(5) PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.
在世界各地的乳制品工厂中，发现了三类基因不同的乳酸乳球菌噬菌体，即936、c2和P335种。多重聚合酶链反应（PCR）方法经过调整，可在单一反应中检测乳清样品或噬菌体裂解物中这些种类的存在。基于它们基因组的保守区域设计了三组引物，每种噬菌体一组。以主要衣壳蛋白基因（mcp）为靶点构建了c2特异性引物。测定了三种噬菌体（eb1、Q38和Q44）的mcp序列，并与数据库中现有的两种噬菌体（c2和bIL67）的mcp序列进行比较。在五个mcp基因中发现了86.4%的同源性。选择唯一的主要结构蛋白（msp）基因作为检测936相关噬菌体的靶点。还确定了三种噬菌体（p2、Q7和Q11）的msp序列，并与噬菌体sk1、bIL170和F4-1的现有数据进行匹配。对六个msp基因的比较显示出82.2%的同源性。在P335样噬菌体的结构蛋白中观察到高度的基因组多样性，这表明该物种内乳球菌噬菌体的分类应予以修订。然而，我们在从六个国家分离出的10个P335样噬菌体中鉴定出一个共同的基因组区域。该区域对应于噬菌体r1t的orfF17 - orf18和Tuc2009的orf20 - orf21，并对另外三种P335噬菌体（Q30、P270和ul40）进行了测序。在五个噬菌体的739个碱基对区域内发现了93.4%的同源性。多重PCR方法在乳清中的检测限为10⁴至10⁷噬菌斑形成单位/毫升，经过额外的噬菌体浓缩步骤后检测限为10³至10⁵噬菌斑形成单位/毫升。该方法还可用于检测乳清粉中的噬菌体DNA，也可能检测细菌基因组中的原噬菌体或缺陷噬菌体。
