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Park, Hwang, Ahn, and Nam: Evaluation of Multiplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Sexually Transmitted Infections Using Swab Specimen
Original Article

J Bacteriol Virol 2020;50(1):44-54.

Published online: 9 March 2020

DOI: https://doi.org/10.4167/jbv.2020.50.1.044

Evaluation of Multiplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Sexually Transmitted Infections Using Swab Specimen

Sun-Hwa Park1, 2, Kyung-Ah Hwang2, Ji Hoon Ahn2, Jae-Hwan Nam1, ∗

1Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, Gyeonggi-do, 420-743, Korea

2Department of Research and Development, Genetree Research, Seoul, Korea

Corresponding Jae-Hwan Nam, PhD. Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, Gyeonggi-do 14662, Republic of Korea Phone: +82-2-2164-4917 Fax: +82-2-2164-4917 E-mail: jhnam@catholic.ac.kr

Received 28 November 201919 February 2020       Accepted 21 February 2020

Copyright © 2020 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Sexually transmitted infections (STIs) are caused by the spread of pathogens via sexual activity and can cause serious complications if left untreated, regardless of their symptoms. Therefore, early diagnosis of STI is important, and molecular diagnostic methods for rapid detection and monitoring are needed. In this study, we evaluated a multiplex polymerase chain reaction (PCR) kit for simultaneously detecting 13 different bacterial, fungal, and viral microorganisms that cause STIs. The kit performance was evaluated for its sensitivity, lot-to-lot variation, and interference in detecting different pathogens. Additionally, its clinical usefulness was evaluated by estimating its sensitivity and specificity for clinical samples. The limit of detection (LOD) was 0.021–50.104 copies for each pathogen. In the tests of lot-to-lot, 100% of positive samples were detected at low concentrations and negative samples all showed negative results. This result confirms that there is no the variation of lot-to-lot. In the test for interference between pathogens, the efficiency of amplification for each pathogen was not significantly reduced and no nonspecific amplification product was formed. We tested 322 vaginal swab samples using the multiplex PCR kit and confirmed that its clinical sensitivity and specificity were 100% for all pathogens. This multiplex PCR kit can be used widely for rapid diagnosis and monitoring of STIs.

Keywords: sexually transmitted infection, multiplex PCR, evaluation

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Fig. 1.
DNA amplification by multiplex PCR (E kit) in clinical specimens
The band size of each amplification product was checked against the positive control. Lanes #1 and 20: Ladder to distinguish amplification bands. Lane #2: positive control for 7 pathogens amplified by Primer Set 2, Lane #10: positive control for 6 pathogens amplified by Primer Set 1, Lanes #3-9 and #11-17: PCR results from clinical samples, Lanes #18 and 19: negative control (distilled water).
jbv-50-44f1.tif
Table 1.
Pathogen-specific regions amplified
Organism (Accession) Target Gene Amplicon Size (bp) position
CA (MN743895) internal transcribed spacer 187 40-226
CT (MG733347) major outer membrane protein 117 5-121
GV (JQ354967) CPN60 407 4-410
HD (AY603047) hemoglobin receptor precursor 670 1685-2354
HSV-1 (EF177455) glycoprotein B (UL27) 330 1279-1608
HSV-2 (KF588415) glycoprotein B (UL27) 570 1042-1611
MG (KP318824) adhesion (mgpB) 248 231-478
MH (KU726667) 16S rRNA 335 91-425
NG (AJ010733) porA pseudogene 175 657-731
TP (M88769) 47-kilodalton antigen gene 252 561-812
TV (L23861) G7 hypothetical protein 690 349-1038
UP (AF085733) UreB 119 632-750
UU (AF085721) UreA 431 302-732

Abbreviations: CA, Candida albicans; CT, Chlamydia trachomatis; GV, Gardnerella vaginalis; HD, Haemophilus ducreyi; HSV-1, Herpes simplex 1; HSV-2, Herpes simplex 2; MG, Mycoplasma genitalium; MH, Mycoplasma hominis; NG, Neisseria gonorrhoeae; TP, Treponema pallidum; TV, Trichomonas vaginalis; UP, Ureaplasma parvum; UU, Ureaplasma urealyticum.

Table 2.
Limit of detection of E kit multiplex PCR
Pathogen DNA Concentrations (copies/μ L) LOD
CA 0.01-10 4.918
CT 0.01-10 1.212
GV 0.01-10 4.918
HD 1-1000 50.104
HSV-1 0.1-100 17.077
HSV-2 0.1-100 16.231
MG 0.1-100 4.3
MH 0.1-100 2.636
NG 0.01-10 1.708
TP 0.1-100 2.636
TV 0.001-1 0.021
UP 0.1-100 3.071
UU 0.1-100 33.577

Abbreviations: LOD, limit of detection.

Table 3.
Lot-to-lot variation for each pathogen
Pathogen Concentration No. of Positive reaction
Lot 1 Lot 2 Lot 3
CA 10 × LOD 20 20 20
1000 × LOD 20 20 20
CT 10 × LOD 20 20 20
1000 × LOD 20 20 20
GV 10 × LOD 20 20 20
1000 × LOD 20 20 20
HD 10 × LOD 20 20 20
1000 × LOD 20 20 20
HSV-1 10 × LOD 20 20 20
1000 × LOD 20 20 20
HSV-2 10 × LOD 20 20 20
1000 × LOD 20 20 20
MG 10 × LOD 20 20 20
1000 × LOD 20 20 20
MH 10 × LOD 20 20 20
1000 × LOD 20 20 20
NG 10 × LOD 20 20 20
1000 × LOD 20 20 20
TP 10 × LOD 20 20 20
1000 × LOD 20 20 20
TV 10 × LOD 20 20 20
1000 × LOD 20 20 20
UP 10 × LOD 20 20 20
1000 × LOD 20 20 20
UU 10 × LOD 20 20 20
1000 × LOD 20 20 20
Distilled water   0 0 0

Abbreviations: LOD, limit of detection.

Table 4.
Test for interference between CT and NG, HSV-1 and HSV-2
Bacterial species (concentration) NG (10 × LOD) NG (100 × LOD) NG (1000 × LOD)
CT (10 × LOD) NG-positive NG-positive NG-positive
CT-positive CT-positive CT-weakly positive
CT (100 × LOD) NG-positive NG-positive NG-positive
CT-positive CT-positive CT-positive
CT (1000 × LOD) NG-weakly positive NG-positive NG-positive
CT-positive CT-positive CT-positive
virus species (concentration) HSV-1 (10 × LOD) HSV-1 (100 × LOD) HSV-1 (1000 × LOD)
HSV-2 (10 × LOD) HSV-1-positive HSV-1-positive HSV-1-positive
  HSV-2-positive HSV-2-positive HSV-2-positive
HSV-2 (100 × LOD) HSV-1-positive HSV-1-positive HSV-1-positive
  HSV-2-positive HSV-2-positive HSV-2-positive
HSV-2 (1000 × LOD) HSV-1-positive HSV-1-positive HSV-1-positive
  HSV-2-positive HSV-2-positive HSV-2-positive
Table 5.
Sensitivities and specificities of E kit in 322 swab specimens
Control PCR & sequencing Results Sensitivity (%) (95% CI) Specificity (%) (95% CI) PPV (%) NPV (%)
Organism Positive Negative
CA 44 278 100 (96.61–100) 100 (99.36–100) 100 100
CT 38 284 100.0 (95.01–100) 100.0 (99.39–100) 100 100
GV 138 184 100 (98.87–100) 100 (99.00–100) 100 100
HD 0 322 NA NA 100 100
HSV-1 17 305 100 (94.31–100) 100 (99.40–100) 100 100
HSV-2 24 298 100 (94.22–100) 100 (99.40–100) 100 100
MH 65 257 100 (97.75–100) 100 (99.29–100) 100 100
MG 33 289 100 (94.72–100) 100 (99.40–100) 100 100
NG 28 294 100.0 (94.40–100) 100.0 (99.40–100) 100 100
TP 1 321 100 (93.02–100) 100 (99.41–100) 100 100
TV 16 306 100 (94.22–100) 100 (99.40–100) 100 100
UP 74 248 100 (98.68–100) 100 (99.09–100) 100 100
UU 60 262 100 (96.79–100) 100 (99.35–100) 100 100

Abbreviations: PCR, polymerase chain reaction; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.

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