1887

Journal of Medical Microbiology logo

Volume 64, Issue 9

Detection of the oomycete by real-time PCR targeting the gene coding for exo-1,3-β-glucanase Free

Abstract

Pythiosis is a life-threatening infectious disease caused by . Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of . Failure to detect by PCR amplification using the rDNA-specific primers has been reported. , encoding an exo-1,3-β-glucanase, is an alternative, novel and efficient target for identification of by conventional PCR. In this study, we aimed to develop a real-time (RT)-PCR approach targeting and compare its performance with conventional PCR for the detection of . Both conventional and RT-PCR assays were positive for all 35 strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1 × 10 ng, respectively. In conclusion, the RT-PCR assay retained 100 % sensitivity and 100 % specificity for detection of . It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of .

  • Received:
  • Accepted:
  • Published Online:
© 2015 The Authors
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000117
2015-09-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/jmm/64/9/971.html?itemId=/content/journal/jmm/10.1099/jmm.0.000117&mimeType=html&fmt=ahah

References

  1. Badenoch P. R., Coster D. J., Wetherall B. L., Brettig H. T., Rozenbilds M. A., Drenth A., Wagels G. 2001; Pythium insidiosum keratitis confirmed by DNA sequence analysis. Br J Ophthalmol 85:496 [View Article][PubMed]
     [Google Scholar]
  2. Bosco S., de M G., Bagagli E., Araújo J. P. Jr, Candeias J. M. G., de Franco M. F., Alencar Marques M. E., Mendoza L., de Camargo R. P., Alencar Marques S. 2005; Human pythiosis, Brazil. Emerg Infect Dis 11:715–718 [View Article][PubMed]
     [Google Scholar]
  3. Brown N. P., Leroy C., Sander C. 1998; MView: a web-compatible database search or multiple alignment viewer. Bioinformatics 14:380–381 [View Article][PubMed]
     [Google Scholar]
  4. Cankar K., Stebih D., Dreo T., Zel J., Gruden K. 2006; Critical points of DNA quantification by real-time PCR - effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. BMC Biotechnol 6:37 [View Article][PubMed]
     [Google Scholar]
  5. Chaiprasert A., Samerpitak K., Wanachiwanawin W., Thasnakorn P. 1990; Induction of zoospore formation in Thai isolates of Pythium insidiosum. Mycoses 33:317–323[PubMed]
     [Google Scholar]
  6. Chareonsirisuthigul T., Khositnithikul R., Intaramat A., Inkomlue R., Sriwanichrak K., Piromsontikorn S., Kitiwanwanich S., Lowhnoo T., Yingyong W., other authors. 2013; Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis. Diagn Microbiol Infect Dis 76:42–45 [View Article][PubMed]
     [Google Scholar]
  7. Grooters A. M., Gee M. K. 2002; Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum. J Vet Intern Med 16:147–152 [View Article][PubMed]
     [Google Scholar]
  8. Jindayok T., Piromsontikorn S., Srimuang S., Khupulsup K., Krajaejun T. 2009; Hemagglutination test for rapid serodiagnosis of human pythiosis. Clin Vaccine Immunol 16:1047–1051 [View Article][PubMed]
     [Google Scholar]
  9. Karlen Y., McNair A., Perseguers S., Mazza C., Mermod N. 2007; Statistical significance of quantitative PCR. BMC Bioinformatics 8:131 [View Article][PubMed]
     [Google Scholar]
  10. Keeratijarut A., Karnsombut P., Aroonroch R., Srimuang S., Sangruchi T., Sansopha L., Mootsikapun P., Larbcharoensub N., Krajaejun T. 2009; Evaluation of an in-house immunoperoxidase staining assay for histodiagnosis of human pythiosis. Southeast Asian J Trop Med Public Health 40:1298–1305[PubMed]
     [Google Scholar]
  11. Keeratijarut A., Lohnoo T., Yingyong W., Nampoon U., Lerksuthirat T., Onpaew P., Chongtrakool P., Krajaejun T. 2014; PCR amplification of a putative gene for exo-1, 3-β-glucanase to identify the pathogenic oomycete Pythium insidiosum. Asian Biomed 8:637–644
     [Google Scholar]
  12. Krajaejun T., Sathapatayavongs B., Pracharktam R., Nitiyanant P., Leelachaikul P., Wanachiwanawin W., Chaiprasert A., Assanasen P., Saipetch M., other authors. 2006a; Clinical and epidemiological analyses of human pythiosis in Thailand. Clin Infect Dis 43:569–576 [View Article][PubMed]
     [Google Scholar]
  13. Krajaejun T., Kunakorn M., Pracharktam R., Chongtrakool P., Sathapatayavongs B., Chaiprasert A., Vanittanakom N., Chindamporn A., Mootsikapun P. 2006b; Identification of a novel 74-kiloDalton immunodominant antigen of Pythium insidiosum recognized by sera from human patients with pythiosis. J Clin Microbiol 44:1674–1680 [View Article][PubMed]
     [Google Scholar]
  14. Krajaejun T., Imkhieo S., Intaramat A., Ratanabanangkoon K. 2009; Development of an immunochromatographic test for rapid serodiagnosis of human pythiosis. Clin Vaccine Immunol 16:506–509 [View Article][PubMed]
     [Google Scholar]
  15. Krajaejun T., Keeratijarut A., Sriwanichrak K., Lowhnoo T., Rujirawat T., Petchthong T., Yingyong W., Kalambaheti T., Smittipat N., other authors. 2010; The 74-kilodalton immunodominant antigen of the pathogenic oomycete Pythium insidiosum is a putative exo-1,3-β-glucanase. Clin Vaccine Immunol 17:1203–1210 [View Article][PubMed]
     [Google Scholar]
  16. Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F., Wallace I. M., Wilm A., other authors. 2007; clustal w clustal x version 2.0. Bioinformatics 23:2947–2948 [View Article][PubMed]
     [Google Scholar]
  17. Lohnoo T., Jongruja N., Rujirawat T., Yingyon W., Lerksuthirat T., Nampoon U., Kumsang Y., Onpaew P., Chongtrakool P., other authors. 2014; Efficiency comparison of three methods for extracting genomic DNA of the pathogenic oomycete Pythium insidiosum. J Med Assoc Thai 97:342–348[PubMed]
     [Google Scholar]
  18. Mackay I. M. 2004; Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 10:190–212 [View Article][PubMed]
     [Google Scholar]
  19. Mendoza L., Ajello L., McGinnis M. R. 1996; Infection caused by the oomycetous pathogen Pythium insidiosum. J Mycol Med 6:151–164
     [Google Scholar]
  20. Reis J. L. Jr, de Carvalho E. C. Q., Nogueira R. H. G., Lemos L. S., Mendoza L. 2003; Disseminated pythiosis in three horses. Vet Microbiol 96:289–295 [View Article][PubMed]
     [Google Scholar]
  21. Rivierre C., Laprie C., Guiard-Marigny O., Bergeaud P., Berthelemy M., Guillot J. 2005; Pythiosis in Africa. Emerg Infect Dis 11:479–481[PubMed]
     [Google Scholar]
  22. Rujirawat T., Patumcharoenpol P., Lohnoo T., Yingyong W., Lerksuthirat T., Tangphatsornruang S., Suriyaphol P., Grenville-Briggs L. J., Garg G., other authors. 2015; Draft genome sequence of the pathogenic oomycete Pythium insidiosum strain Pi-S, isolated from a patient with pythiosis. Genome Announc 3:e00574–e00515 [View Article][PubMed]
     [Google Scholar]
  23. Stajich J. E., Harris T., Brunk B. P., Brestelli J., Fischer S., Harb O. S., Kissinger J. C., Li W., Nayak V., other authors. 2012; FungiDB: an integrated functional genomics database for fungi. Nucleic Acids Res 40:(D1)D675–D681 [View Article][PubMed]
     [Google Scholar]
  24. Supabandhu J., Fisher M. C., Mendoza L., Vanittanakom N. 2008; Isolation and identification of the human pathogen Pythium insidiosum from environmental samples collected in Thai agricultural areas. Med Mycol 46:41–52 [View Article][PubMed]
     [Google Scholar]
  25. Vanittanakom N., Supabandhu J., Khamwan C., Praparattanapan J., Thirach S., Prasertwitayakij N., Louthrenoo W., Chiewchanvit S., Tananuvat N. 2004; Identification of emerging human-pathogenic Pythium insidiosum by serological and molecular assay-based methods. J Clin Microbiol 42:3970–3974 [View Article][PubMed]
     [Google Scholar]
  26. Wilhelm J., Pingoud A., Hahn M. 2003; Real-time PCR-based method for the estimation of genome sizes. Nucleic Acids Res 31:e56 [View Article][PubMed]
     [Google Scholar]
  27. Ye J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S., Madden T. L. 2012; Primer-blast: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134 [View Article][PubMed]
     [Google Scholar]
  28. Znajda N. R., Grooters A. M., Marsella R. 2002; PCR-based detection of Pythium and Lagendium DNA in frozen and ethanol-fixed animal tissues. Vet Dermatol 13:187–194 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000117
Loading
Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-β-glucanase
J. Med. Microbiol. 64, 971 (2015); https://doi.org/10.1099/jmm.0.000117
/content/journal/jmm/10.1099/jmm.0.000117
/content/journal/jmm/10.1099/jmm.0.000117
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error