Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

被引：17

作者：

Aguilera-Arreola M.G. ^{[1
]}

González-Cardel A.M. ^{[1
,4
]}

Tenorio A.M. ^{[2
]}

Curiel-Quesada E. ^{[3
]}

Castro-Escarpulli G. ^{[1
]}

机构：

[1] Medical Bacteriology, Department of Microbiology, Escuela Nacional de Ciencias Biológicas of Instituto Politécnico Nacional (ENCB-IPN), Del. Miguel Hidalgo CP 11340, Mexico DF, Esq.Prol.Carpio y Plan de Ayala s/n Col.Santo Tomas

[2] Laboratory of Biotechnology and Genomic Bioinformatics, Department of Biochemistry, Escuela Nacional de Ciencias Biológicas of Instituto Politécnico Nacional (ENCB-IPN), Del. Miguel Hidalgo CP 11340, Mexico DF, Esq.Prol.Carpio y Plan de Ayala s/n Col. Sant

[3] Genetic Engineering, Department of Biochemistry, Escuela Nacional de Ciencias Biológicas of Instituto Politécnico Nacional (ENCB-IPN), Del. Miguel Hidalgo CP 11340, Mexico DF, Esq.Prol.Carpio y Plan de Ayala s/n Col. SantoTomas

[4] Special Test Laboratory, CMN 20 de Noviembre ISSSTE, Col.Del Valle,CP 03229 Del.Benito Juarez,Mexico DF

来源：

BMC Research Notes | / 7卷 / 1期

关键词：

16S rDNA genes; Cervicitis; Detection; Multiplex PCR;

D O I：

10.1186/1756-0500-7-433

中图分类号：

学科分类号：

摘要：

Background: Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods. The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results: Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions: The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. © 2014 Aguilera-Arreola et al.; licensee BioMed Central Ltd.

引用

共 21 条

[1]

Rodrigues M.M., Fernandes P., Haddad J.P., Paiva M.C., Souza M.C., Andrade T.C., Fernandes A.P., Frequency of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma species in cervical samples, J Obstet Gynaecol, 31, pp. 237-241, (2011)

[2]

Marrazo J.M., Cervicitis, Current Diagnosis & Treatment of Sexually Transmitted Diseases, pp. 60-65, (2007)

[3]

Darville T., Recognition and treatment of chlamydial infections from birth to adolescence, Adv Exp Med Biol, 764, pp. 109-122, (2013)

[4]

Sethi S., Golparian D., Bala M., Dorji D., Ibrahim M., Jabeen K., Unemo M., Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae isolates from India, Pakistan and Bhutan in 2007-2011, BMC Infect Dis, 13, (2013)

[5]

Colson P., Gouriet F., Badiaga S., Tamalet C., Stein A., Raoult D., Real-time laboratory surveillance of sexually-transmissible infections in Marseille University hospitals reveals rise of gonorrhoea, syphilis and human immunodeficiency virus seroconversions in 2012, Euro Surveill, 18, (2013)

[6]

Patel M.A., Nyirjesy P., Role of Mycoplasma and ureaplasma species in female lower genital tract infections, Curr Infect Dis Rep, 12, pp. 417-422, (2010)

[7]

Whiley D.M., Tapsall J.W., Sloots T.P., Nucleic acid amplification testing for Neisseria gonorrhoeae: An ongoing challenge, Journal of Molecular Diagnostics, 8, 1, pp. 3-15, (2006)

[8]

Geisler M.W., Stamm W.E., Genital Chlamydial Infections, Current Diagnosis & Treatment of Sexually Transmitted Diseases, pp. 75-83, (2007)

[9]

De Haro-Cruz M.J., Deleon-Rodriguez I., Escobedo-Guerra M.R., Lopez-Hurtado M., Arteaga-Troncoso G., Ortiz-Ibarra F.J., Guerra-Infante F.M., Genotyping of Chlamydia trachomatis from endocervical specimens of infertile Mexican women, Enferm Infecc Microbiol Clin, 29, pp. 102-108, (2011)

[10]

Hernandez-Martinez F., Hernandez-Garcia J.A., Martinez-Pena M.D., Muniz-Becerril B.L., Hernandez-Cortez C., Castro-Escarpulli G., Aguilera-Arreola M.G., Aetiology and frequency of cervico-vaginal infections among Mexican women, Afr J Microbiol Res, 7, pp. 27-34, (2013)

← 1 2 3 →