Development of New Modified Simple Polymerase Chain Reaction and Real-time Polymerase Chain Reaction for the Identification of Iranian Brucella abortus Strains

Document Type: Original Articles

Authors

1 Department of Brucella, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

2 Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Abstract

Brucellosis is primarily a worldwide zoonotic disease caused by Brucella species. The genus Brucella contains highly infectious species that are classified as biological threat agents. In this regard, the identification of Brucella can be a time-consuming and labor-intensive process posing a real risk of laboratory-acquired infection to the laboratory staff. This study aimed to present a novel conventional and real-time polymerase chain reaction (PCR) assay for the identification of Brucella abortus strains. Regarding this, two primers (bru ab2) were designed based on the unique loci encoding autotransporter-associated beta strand repeat-containing protein (ID:YP00113760). A total of 56 Brucella strains (e.g., reference, vaccinal, and field isolates) and Yersinia enterocolitica, as a non-Brucella isolate, were evaluated in conventional and real-time PCR systems. The results of the study indicated that 0.4 ng and 400 FG of genomic DNA of B. abortus strains can be detected by conventional and real-time PCR, respectively. The primers, bru ab2, were suitable for both PCR methods. Both methods were specific for the detection of all strains of the bacterium; however, real-time PCR assay was 1000-fold more sensitive than the conventional PCR method. Therefore, this new detection system could be a suitable selective modified method for the accurate identification of all B. abortus strains.

Keywords

Main Subjects


Article Title [French]

Développement d'une Nouvelle Réaction en Chaîne de Polymérase Simple Modifiée et d'une Réaction en Chaîne de Polymérase en Temps Réel pour l'identification des Souches Iraniennes de Brucella abortus

Abstract [French]

La brucellose est principalement une maladie zoonotique dans le monde entier causée par l'espèce Brucella. Le genre Brucella contient des espèces hautement infectieuses classées comme agents de menace biologique. À cet égard, l'identification de Brucella peut être un processus long et exigeant en main-d'œuvre, présentant un risque réel d'infection acquise en laboratoire pour le personnel de laboratoire. Cette étude visait à présenter un nouveau test de réaction en chaîne par polymérase (PCR) classique et en temps réel pour l'identification des souches de Brucella abortus. À cet égard, deux amorces (bru ab2) ont été conçues sur la base des locus uniques codant pour une protéine contenant une répétition de brin bêta associée à un autotransporteur (ID: YP00113760). Un total de 56 souches de Brucella (par exemple, des isolats de référence, vaccinal et de terrain) et Yersinia enterocolitica, en tant qu'isolât non-Brucella, ont été évalués dans des systèmes de PCR conventionnels et en temps réel. Les résultats de l’étude ont indiqué que l’ADN génomique des souches de B. abortus à 0,4 ng et 400 FG peut être détecté par PCR conventionnelle et en temps réel, respectivement. Les amorces, bru ab2, convenaient aux deux méthodes de PCR. Les deux méthodes étaient spécifiques à la détection de toutes les souches de la bactérie; cependant, le test de PCR en temps réel était 1000 fois plus sensible que le procédé PCR classique. Par conséquent, ce nouveau système de détection pourrait constituer une méthode modifiée sélective et appropriée pour l'identification précise de toutes les souches de B. abortus.

Keywords [French]

  • Brucella abortus
  • identification
  • PCR
  • PCR en Temps Réel
Adone, R., Pasquali, P., 2013. Epidemiosurveillance of brucellosis. Rev Sci Tech 32, 1, 199-205.

Al Dahouk, S., Nockler, K., Scholz, H.C., Pfeffer, M., Neubauer, H., Tomaso, H., 2007. Evaluation of genus-specific and species-specific real-time PCR assays for the identification of Brucella spp. Clin Chem Lab Med 45, 11, 1464-1470.

Alişkan, H., 2008. The value of culture and serological methods in the diagnosis of human brucellosis. Mikrobiyol Bul 45, 1, 185-195

Chain, P.S., Comerci, D.J., Tolmasky, M.E., Larimer, F.W., Malfatti, S.A., Vergez, L.M., et al., 2005. Whole-genome analyses of speciation events in pathogenic Brucellae. Infect Immun 73, 12, 8353-8361.

Davis, C.E., Troy, S.B., 2005. Brucellosis. N Engl J Med 353, 10, 1071-1072.

De, B.K., Stauffer, L., Koylass, M.S., Sharp, S.E., Gee, J.E., Helsel, L.O., et al., 2008. Novel Brucella strain (BO1) associated with a prosthetic breast implant infection. J Clin Microbiol 46, 1, 43-49.                 

Dean, A.S., Crump, L., Greter, H., Hattendorf, J., Schelling, E., Zinsstag, J., 2012. Clinical manifestations of human brucellosis: a systematic review and meta-analysis. PLoS Negl Trop Dis 6, 12, e1929.

Ficht, T., 2010. Brucella taxonomy and evolution. Future Microbiol 5, 6, 859-866.

Filippov, A.A., Sergueev, K.V., Nikolich, M.P., 2013. Bacteriophages against biothreat bacteria: diagnostic, environmental and therapeutic applications. J Bioterr Biodef 3, 010.

Godfroid, J., Scholz, H.C., Barbier, T., Nicolas, C., Wattiau, P., Fretin, D., et al., 2011. Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Prev Vet Med 102, 2, 118-131.

Gopaul, K.K., Sells, J., Lee, R., Beckstrom-Sternberg, S.M., Foster, J.T., Whatmore, A.M., 2014. Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species. BMC Res Notes 7, 1, 903.

Lista, F., Reubsaet, F.A., De Santis, R., Parchen, R.R., de Jong, A.L., Kieboom, J., et al., 2011. Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS. BMC Microbiol 11, 267.

Molavi, M.A., Sajjadi, H.S., Nejatizade, A.A., 2014. Effective methods for appropriate diagnosis of brucellosis in humans and animals (review article). Online J Anim Feed Res (OJAFR) 4, 3, 60-66.

Najafi, N., Ghassemian, R., Davoody, A.R., Tayebi, A., 2011. An unusual complication of a common endemic disease: clinical and laboratory aspects of patients with brucella epididymoorchitis in the north of Iran. BMC Res Notes 4, 286.

Newby, D.T., Hadfield, T.L., Roberto, F.F., 2003. Real-time PCR detection of Brucella abortus: a comparative study of SYBR green I, 5'-exonuclease, and hybridization probe assays. Appl Environ Microbiol 69, 8, 4753-4759.

O'Grady, D., Byrne, W., Kelleher, P., O'Callaghan, H., Kenny, K., Heneghan, T., et al., 2014. A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle. Vet J 199, 3, 370-375.

Pappas, G., Akritidis, N., Bosilkovski, M., Tsianos, E., 2005. Brucellosis. N Engl J Med 352, 22, 2325-2336.

Probert, W.S., Schrader, K.N., Khuong, N.Y., Bystrom, S.L., Graves, M.H., 2004. Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J Clin Microbiol 42, 3, 1290-1293.

Redkar, R., Rose, S., Bricker, B., DelVecchio, V., 2001. Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis. Mol Cell Probes 15, 1, 43-52.

Santis, R.d., Ciammaruconi, A., Pomponi, A., Fillo, S., Lista, F., 2011. Brucella: molecular diagnostic techniques in response to bioterrorism threat. J Bioter Biodef 5, S2, 1-8.

Scholz, H.C., Hubalek, Z., Sedlacek, I., Vergnaud, G., Tomaso, H., Al Dahouk, S., et al., 2008. Brucella microti sp. nov., isolated from the common vole Microtus arvalis. Int J Syst Evol Microbiol 58, Pt2, 375-382.

Scholz, H.C., Vergnaud, G., 2013. Molecular characterisation of Brucella species. Rev Sci Tech 32, 1, 149-162.

Smirnova, E., Vasin, A., Sandybayev, N., Klotchenko, S., Plotnikova, M., Chervyakova, O., et al., 2013. Current Methods of Human and Animal Brucellosis Diagnostics. Adv Infect Dis 3, 3, 177-184.

Sohn, A.H., Probert, W.S., Glaser, C.A., Gupta, N., Bollen, A.W., Wong, J.D., et al., 2003. Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp. Emerg Infect Dis 9, 4, 485-488.

Ica, T., Aydin, F., Gümüşsoy, K., Perçin, D., Bulent, S., Ahmet, et al., 2012. Conventional and Molecular Biotyping of Brucella Strains Isolated from Cattle, Sheep and Human in Kayseri, Turkey. Vet Fak Derg 59, 259-264.

Tiller, R.V., Gee, J.E., Lonsway, D.R., Gribble, S., Bell, S.C., Jennison, V., et al., 2010. Identification of an unusual Brucella strain (BO2) from a lung biopsy in a 52 year-old patient with chronic destructive pneumonia. BMC Microbiol 10, 1, 23.

Troy, S.B., Rickman, L.S., Davis, C.E., 2005. Brucellosis in San Diego: epidemiology and species-related differences in acute clinical presentations. Medicine (Baltimore) 84, 3, 174-187.

 

 

 

 

Ullah, R.W., Waqas, M.Y., Muhammad Ali Shah, M.A., 2014. Epidemiology of Bovine Brucellosis-a review of literature. Veterinaria 1, 16-19.

Winchell, J.M., Wolff, B.J., Tiller, R., Bowen, M.D., Hoffmaster, A.R., 2010. Rapid identification and discrimination of Brucella isolates by use of real-time PCR and high-resolution melt analysis. J Clin Microbiol 48, 3, 697-702.