Previous Article | Next Article 
Journal of Clinical Microbiology, June 2009, p. 1871-1877, Vol. 47, No. 6
0095-1137/09/$08.00+0 doi:10.1128/JCM.00120-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Simultaneous Identification of 14 Genital Microorganisms in Urine by Use of a Multiplex PCR-Based Reverse Line Blot Assay
,
Michelle L. Mckechnie,1,2
Richard Hillman,1
Deborah Couldwell,3
Fanrong Kong,2
Eleanor Freedman,4
Hui Wang,2,5 and
Gwendolyn L. Gilbert2*
Sexually Transmitted Infections Research Centre (STIRC), University of Sydney, Marian Villa, Westmead Hospital, New South Wales 2145, Australia,1 Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales 2145, Australia,2 Parramatta Sexual Health Clinic, Level 2, Jeffery House, 158 Marsden St., Parramatta, New South Wales 2145, Australia,3 Sydney Sexual Health Centre, Sydney Hospital and Eye Hospital, Macquarie Street, Sydney, New South Wales 2000, Australia,4 Research Laboratory for Infectious Skin Diseases, Department of Dermatology, Wuhan First Hospital, Wuhan 430022, People's Republic of China5
Received 21 January 2009/ Returned for modification 27 February 2009/ Accepted 31 March 2009
The aim of this study was to develop and evaluate a sensitive method for the simultaneous identification of 14 urogenital potential pathogens. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to detect 14 urogenital pathogens or putative pathogens, namely Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus type 1 (HSV1) and HSV2, N. meningitidis, Mycoplasma hominis, M. genitalium, and adenovirus, using two species-specific primer pairs and probes for each. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism and was found to be both sensitive and specific. The limits of detection for the mPCR/RLB assay varied among the 14 target organisms from 4.2 x 10–1 to 7.0 x 10–11 ng/µl of genomic DNA. There were no cross-reactions among any of the probes. This method was used to test 529 first-voided urine specimens from male patients with and without urethritis attending two Sydney sexual health clinics. One or more target species were detected in 193 (36%) subjects. Of 233 positive results, overall 216 (93%) were concordant between mPCR/RLB and a comparator method (culture and/or species-specific PCR), 9 were positive only by mPCR/RLB, and 8 were positive only by the comparator method. The mPCR/RLB method was an accurate, convenient, and inexpensive method for the detection of multiple potential pathogens in first-voided urine specimens from men.
* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Darcy Road, Westmead, New South Wales 2145, Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: l.gilbert{at}usyd.edu.au
Published ahead of print on 8 April 2009.
Supplemental material for this article may be found at http://jcm.asm.org/.
Journal of Clinical Microbiology, June 2009, p. 1871-1877, Vol. 47, No. 6
0095-1137/09/$08.00+0 doi:10.1128/JCM.00120-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»