Almeida Sintia, Dorneles Elaine M S, Diniz Carlos, Abreu Vinícius, Sousa Cassiana, Alves Jorianne, Carneiro Adriana, Bagano Priscilla, Spier Sharon, Barh Debmalya, Lage Andrey P, Figueiredo Henrique, Azevedo Vasco
Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Escola de Veterinária, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
BMC Vet Res. 2017 Sep 25;13(1):290. doi: 10.1186/s12917-017-1210-5.
Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis.
In the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level.
A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410, were compared to the results of nitrate reductase identification by biochemical test. The McNemar's Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16-6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90-0.98)].
The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.
伪结核棒状杆菌分为两个生物变种，硝酸盐阴性的绵羊生物变种，它是小反刍动物干酪性淋巴结炎的病原体，以及硝酸盐阳性的马生物变种，它会导致马属动物出现脓肿和溃疡性淋巴管炎。本研究的目的是开发一种四重PCR检测方法，用于同时检测伪结核棒状杆菌并进行生物变种分型。
在本研究中，利用伪结核棒状杆菌菌株的基因组来鉴定参与硝酸盐还原途径的基因，以改进一种用于物种鉴定的三引物多重PCR检测方法。将硝酸盐还原酶基因（narG）与16S、rpoB和pld基因一起纳入PCR检测，以提高多重PCR在生物变种水平上的诊断能力。
开发了一种用于伪结核棒状杆菌物种和生物变种鉴定的新型四重PCR检测方法。对348株菌株进行四重PCR检测的结果，与346株先前已充分鉴定的来自不同宿主（山羊、绵羊、马、牛、水牛、美洲驼和人类）的伪结核棒状杆菌临床分离株、疫苗株1002和模式菌株ATCC 19410的结果进行了比较，并与通过生化试验进行硝酸盐还原酶鉴定的结果进行了比较。用于比较两种伪结核棒状杆菌生物变种鉴定方法的McNemar卡方检验显示，四重PCR与硝酸盐生化试验之间无显著差异（P = 0.75）[优势比的95%置信区间（0.
