Development and validation of a multiplex polymerase chain reaction for molecular identification of Salmonella enterica serogroups B, C2, D and E
Keywords:
Salmonella enterica, serogroups, serotyping, molecular typing, multiplex PCR
Abstract
Introduction. The scheme Kauffman-White (KW) for serotyping of Salmonella recognizes 46 O antigens, and 119 H antigens, thereby permitting the characterization of 2,541 serotypes. The serotyping is a useful epidemiological tool in identifying circulating serotypes and to characterize outbreaks. However, the method presents technical limitations, difficulty in interpretation of results and high costs.Objective. A multiplex polymerase chain reaction test (M-PCR) was developed as an alternative method for the identification of serogroups B, C2, D, and E of Salmonella enterica.
Materials and methods. The M-PCR detected Salmonella genes rfbJ of serogroups B and C2 and wzx of serogroups D and E. To standardize the M-PCR, reference strains of Salmonella serogroups were compared. Amplification of invA gender-specific gene of Salmonella was included as internal control of amplification. To validate the test, a blind study was conducted to identify by M-PCR 400 isolates that had been previously characterized by serology.
Results. The M-PCR detected Salmonella serogroups with reproducible results (Kappa index=0.95). The sensitivity of the test was between 98% to 100% and specificity between 96% to 100%.
Conclusions. The polymorphisms in the Salmonella genes rfbJ and wzx permitted the development of a method for molecular typing of Salmonella serogroups that was sensitive, specific and reproducible.
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References
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17. Álvarez J, Sota M, Vivanco AB, Perales I, Cisterna R, Rementeria A, et al. Development of a multiplex PCR technique for detection and epidemiological typing of Salmonella in human clinical samples. J Clin Microbiol. 2004;42:1734-8.
18. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev. 2000;13:559-70.
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21. Curd H, Liu D, Reeves PR. Relationships among the O-antigen gene clusters of Salmonella enterica groups B, D1, D2 and D3. J Bacteriol. 1998;180:1002-7.
22. Hellerqvist CG, Rudén U. The group C2-type modification of the B-Type lipopolysaccharide in a hybrid between Salmonella groups B and C2. Eur J Biochem. 1972;25:96-101.
23. Wang L, Andrianopoulos K, Liu D, Popoff MY, Reeves PR. Extensive variation in the O-Antigen gene cluster within one Salmonella enterica serogroup reveals an unexpected complex history. J Bacteriol. 2002;184:1669-77.
24. Herrera LS, Ramiro R, Arroyo M, Díez R, Usera MA, Echeita MA. Blind comparison of traditional serotyping with three multiplex PCR for identification of Salmonella serotypes. Res Microbiol. 2007;158:122-7.
25. Muñoz N, Firacative C, Realpe ME, Patiño L, Gómez ME, Murcia LM, et al. Informe anual de la vigilancia fenotípica y molecular de Salmonella spp. en Colombia, 2007. Inf Quinc Epidemiol Nac. 2008;13:207-22.
26. Malkawi HI, Gharaibeh R. Rapid and simultaneous identification of two Salmonella enterica serotypes, Enteritidis and Typhimurium from chicken and meat products by multiplex PCR. Biotechnology. 2004;3:44-8.
27. Sánchez MM, Cardona-Castro N. Desarrollo y evaluación de una prueba de reacción en cadena de la polimerasa (PCR), utilizando la secuencia del gen hilA para diagnóstico de fiebre entérica por Salmonella spp. Biomédica. 2004;24:194-9.
2. Brenner FW, Villar RG, Angulo FJ, Tauxe R, Swaminathan B. Salmonella nomenclature. J Clin Microbiol. 2000;38:2465-7.
3. Kauffmann F. The bacteriology of Enterobacteriaceae. Baltimore: Williams & Wilkins; 1966.
4. Fitzgerald C, Gheesling L, Collins M, Fields PI. Sequence analysis of the rfb loci, encoding proteins involved in the biosynthesis of the Salmonella enterica O17 and O18 antigens: serogroup-specific identification by PCR. Appl Environ Microbiol. 2006;72:7949-53.
5. Verma NK, Quigley NB, Reeves PR. O-antigen variation in Salmonella spp rfb gene clusters of three strains. J Bacteriol. 1998;170:103-7.
6. Liu D, Haase AM, Lindqvist L, Lindberg AA, Reeves PR. Glycosyl transferases of O-antigen biosynthesis in Salmonella enterica: identification and characterization of transferase genes of groups B, C2, and E1. J Bacteriol. 1993;175:3408-13.
7. Wyk P, Reeves PR. Identification and sequence of the gene for abequose synthase, which confers antigenic specificity on group B Salmonellae: homology with galactose epimerase. J Bacteriol. 1989;171:5687-93.
8. Liu D, Cole RA, Reeves PR. An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol. 1996;178:2102-7.
9. Mortimer CK, Peters TM, Gharbia SE, Logan MJ, Arnold C. Towards the development of a DNA-sequence based approach to serotyping of Salmonella enterica. BMC Microbiol. 2004;4:31-40.
10. Karami A, Ranjbar R, Ahmadi Z, Safiri Z. Rapid detection of different serovares of Salmonella enterica by multiplex PCR. Iranian J Publ Health. 2007;36:38-42.
11. Soltani MJ, Shahhosseiny MH, Shahbazzadeh D, Karami V, Mirzahoseini H, Mahboudi F, et al. Selective amplification of prt, tyv,and invA genes by multiplex PCR for rapid detection of Salmonella typhi. Iranian J Publ Health. 2005;9:135-8.
12. Lim YH, Hirose K, Izumiya H, Arakawa E, Takahashi H, Terajima J, et al. Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar Typhimurium. Jpn J Infect Dis. 2003;56:151-5.
13. Luk JM, Kongmuang U, Reeves PR, Lindberg AA. Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of Salmonella major serogroups (A, B, C2 and D). J Clin Microbiol. 1993;31:2118-23.
14. Muñoz N, Agudelo CI, Ovalle MV, Realpe MH. Vigilancia en red de la susceptibilidad antimicrobiana y de los serotipos de Salmonella spp., Shigella spp. y Vibrio cholerae O1, 1997-1999. Biomédica. 2000;20:210-7.
15. Haque A, Ahmed J, Qureshi JA. Early detection of typhoid by polimerase chain reaction. Ann Saudi Med. 1999;19:337-40.
16. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques. 1997;23:504-11.
17. Álvarez J, Sota M, Vivanco AB, Perales I, Cisterna R, Rementeria A, et al. Development of a multiplex PCR technique for detection and epidemiological typing of Salmonella in human clinical samples. J Clin Microbiol. 2004;42:1734-8.
18. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev. 2000;13:559-70.
19. Malorny B, Hoorfar J, Bunge C, Helmuth R. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl Environ Microbiol. 2003;69:290-6.
20. Olive DM, Bean P. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol. 1999;37:1661-9.
21. Curd H, Liu D, Reeves PR. Relationships among the O-antigen gene clusters of Salmonella enterica groups B, D1, D2 and D3. J Bacteriol. 1998;180:1002-7.
22. Hellerqvist CG, Rudén U. The group C2-type modification of the B-Type lipopolysaccharide in a hybrid between Salmonella groups B and C2. Eur J Biochem. 1972;25:96-101.
23. Wang L, Andrianopoulos K, Liu D, Popoff MY, Reeves PR. Extensive variation in the O-Antigen gene cluster within one Salmonella enterica serogroup reveals an unexpected complex history. J Bacteriol. 2002;184:1669-77.
24. Herrera LS, Ramiro R, Arroyo M, Díez R, Usera MA, Echeita MA. Blind comparison of traditional serotyping with three multiplex PCR for identification of Salmonella serotypes. Res Microbiol. 2007;158:122-7.
25. Muñoz N, Firacative C, Realpe ME, Patiño L, Gómez ME, Murcia LM, et al. Informe anual de la vigilancia fenotípica y molecular de Salmonella spp. en Colombia, 2007. Inf Quinc Epidemiol Nac. 2008;13:207-22.
26. Malkawi HI, Gharaibeh R. Rapid and simultaneous identification of two Salmonella enterica serotypes, Enteritidis and Typhimurium from chicken and meat products by multiplex PCR. Biotechnology. 2004;3:44-8.
27. Sánchez MM, Cardona-Castro N. Desarrollo y evaluación de una prueba de reacción en cadena de la polimerasa (PCR), utilizando la secuencia del gen hilA para diagnóstico de fiebre entérica por Salmonella spp. Biomédica. 2004;24:194-9.
How to Cite
1.
Cardona-Castro N, Lavalett L, Sánchez MM, Múñoz N, Moreno J. Development and validation of a multiplex polymerase chain reaction for molecular identification of Salmonella enterica serogroups B, C2, D and E. biomedica [Internet]. 2009 Jun. 1 [cited 2024 May 16];29(2):244-52. Available from: https://revistabiomedica.org/index.php/biomedica/article/view/26
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