de Filippis I, de Andrade C F, de Almeida A E C C, Clementino M M, Fernandes C A C M, de Carvalho M L, Vicente A C P
Microbiology Dept., National Institute for Quality Control of Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Lett Appl Microbiol. 2007 Oct;45(4):426-31. doi: 10.1111/j.1472-765X.2007.02205.x.
Rapid characterization of variable region (VR)1 variants of the porA gene among invasive strains is crucial for outbreak management and epidemiology studies. Recent sequence analysis studies in Brazil showed that the VR1 P1.7 and P1.19 variants are highly prevalent, accounting for 68%, of the total number of VR1 variants characterized. The aim of this work is to develop a rapid polymerase chain reaction (PCR)-based method for genosubtyping Neisseria meningitidis by detection of porA variable regions P1.7 and P1.19.
PCR primers for the detection of porA VR1 P1.7 and P1.19 were designed and tested using 198 clinical N. meningitidis isolates that had been previously evaluated by porA sequencing. All 50 strains with VR1 P1.7 and all 65 strains with VR1 P1.19 were positively identified by the respective VR-specific PCR and no false-positive reactions occurred.
VR-specific PCR amplification accurately identified VR P1.7 and P1.19 strains.
To overcome the disadvantages of serosubtyping and sequencing for typing the porA VR1 segment of N. meningitidis, we developed a PCR-based method to rapidly and accurately detect VR1 P1.7 and P1.19 variants. This approach is highly specific and sensitive; moreover it may allow for genotype determination of culture-negative samples.
快速鉴定侵袭性菌株中porA基因可变区(VR)1变异体对于疫情管理和流行病学研究至关重要。巴西最近的序列分析研究表明,VR1 P1.7和P1.19变异体高度流行,占已鉴定的VR1变异体总数的68%。本研究的目的是开发一种基于聚合酶链反应(PCR)的快速方法,通过检测porA可变区P1.7和P1.19对脑膜炎奈瑟菌进行基因分型。
设计了用于检测porA VR1 P1.7和P1.19的PCR引物,并使用198株先前通过porA测序评估的临床脑膜炎奈瑟菌分离株进行了测试。所有50株VR1 P1.7菌株和所有65株VR1 P1.19菌株均通过各自的VR特异性PCR得到阳性鉴定,未出现假阳性反应。
VR特异性PCR扩增准确鉴定了VR P1.7和P1.19菌株。
为克服血清分型和测序在脑膜炎奈瑟菌porA VR1片段分型方面的缺点,我们开发了一种基于PCR的方法来快速准确地检测VR1 P1.7和P1.19变异体。这种方法具有高度的特异性和敏感性;此外,它可能允许对培养阴性样本进行基因型测定。