Chlamydia trachomatisÂ (CT), UreaplasmaÂ urealyticumÂ (UU) and Neisseria gonorrhoeaeÂ (NG) are the most common pathogens of sexually transmitted infectionsÂ (STIs), frequently founded in urogenital infections, and showed a criminal role in increasing the risk of potential adverse outcomes. In this study a multiplex PCRÂ assay for the simultaneous detectionÂ andÂ accurate identification of 3 clinically relevant pathogens of STIs, i.e., CT, NG and UUÂ in a single tubeÂ was developed and evaluated. The limits of detection for the multiplex PCR assay were ~10 copies of DNAs per reaction. This assay has comparable clinical sensitivity to the conventional monoplex real-time PCR assayÂ and considerable potential to be routine molecular diagnostic tool for simultaneous identification of STIs at relatively low cost due to multiplexing

Jiao, Qiancheng

Xie, Yangyang

Ye, Sheng

Yu, Kang

Zheng, Weiji

Zhu, Xiaoling

English

Advanced Emergency Medicine (E-Journal)

Volume 11 | Issue 2 - 11 -Simultaneous Detection of Chlamydia Trachomatis, NeisseriaGonorrhoeae, Ureaplasma Urealyticum by Multiplex PCR-Running Qiancheng Jiao1, Xiaoling Zhu2, Weiji Zheng3, Yangyang Xie3, Kang Yu1*, Sheng Ye3, 4*(Q. Jiao and X. Zhu equally contributed to this work.)1. The 1rd School of Medicine, Wenzhou Medical University, Wenzhou 325035, China.2. School of Nursing, Wenzhou Medical University, Wenzhou 325035, China.3. School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou325035, China.4. Zhejiang YuanchuangMedical Technology Co., Ltd, Wenzhou 325035, China.Abstract:Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU) and Neisseria gonorrhoeae (NG) are the most common pathogens of sexually transmitted infections (STIs), frequently founded in urogenital infections, and showed a criminal role in increasing the risk of potential adverse outcomes. In this study a multiplex PCR assay for the simultaneous detection and accurate identification of 3 clinically relevant pathogens of STIs, i.e., CT, NG and UU in a single tube was developed and evaluated. The limits of detection for the multiplex PCR assay were ~10 copies of DNAs per reaction. This assay has comparable clinical sensitivity to the conventional monoplex real-time PCR assay and considerable potential to be routine molecular diagnostic tool for simultaneous identification of STIs at relatively low cost due to multiplexing.Keywords: Sexually Transmitted Infection; Multiplex PCR; Chlamydia Trachomatis; Ureaplasma Urealyticum; Neisseria GonorrhoeaeIntroductionChlamydia trachomatis (CT), Ureaplasma urealyticum (UU) and Neisseria gonorrhoeae (NG) as the most frequent pathogens ofsexually transmitted infections (STIs) are considered to be the main etiological agents of urogenital disease (Xu et al. 2018; Roy et al.2021). Genital sexually transmitted CT, UU and NG are highly prevalent in women (Liang et al. 2018), increased the risk of potentialadverse pregnancy outcomes, such as preterm birth, low birth weight, spontaneous abortion, perinatal mortality and ophthalmianeonatorum (Vallely et al. 2018), newborn respiratory distress syndrome.To avoid the abuse of antibiotic, guidelines for sexually transmitted infected urethritis suggested that infected with high loadshould be treated rather than routine and treatment of asymptomatic person (Horner et al.2018). Therefore, the accurate determinationof pathogens can avoid the risk of antibiotic resistance and overuse of antibiotics indicated for empirical treatments (Bartoletti et al.2019).Nucleic acid amplification tests performed more specific and sensitive than culture or enzyme immunoassay tests (Marangoni etal. 2012) and increased the finding of case (Pillay et al. 2021). Molecular diagnosis by PCR directly detects pathogen-specific nucleicacid, many of the recently diagnostic methods for STIs employ PCR methods (Muvunyi et al, 2011;Van Der Pol et al. 2017). Severalcommercial multiplex PCR kits have been developed and evaluated (Ursi et al. 2016; Barrientos-Durán et al. 2020).In this survey, multiplex real-time PCR was optimized for accurate identification of CT, UU and NG in a single PCR tube withthe use of three fluorescent probes.Advanced Emergency Medicine- 12 -1. Materials and Methods1.1 Clinical specimensThe DNA samples from the patients were obtained from Hang Zhou KingMed Diagnostics Co., Ltd. Briefly, the swabs wasplaced in a sterile test tube containing 1 mL of sterile normal saline as the transport medium and washed by shock for a while. Theswab was then squeezed dry on the wall and discarded. Then, the samples were stored at -20°C for further treatment.1.2 Primer and probe designAccording to the genome sequences of CT, UU and NG from GeneBank, the software Primer Express was used to design PCRprimers and their corresponding TaqMan MGB probes. Melting temperature (Tm) values and secondary structures were mainlyconsidered. All primers were desalted or HPLC purified grade.1.3 PCR assay designThe PCRAmplification reactions were accomplished in a final volume of 25 μL. The PCR products were run on 2% agarose gelelectrophoresis. Real-time PCR was conducted using theABI7000/7300/7500 Real-Time PCR System and LightCycler 2.0.1.4 PCR sensitivity and specificityUsing the optimized PCR conditions, the sensitivity and specificity of monoplex and multiple PCRs were analyzed with clinicalsamples and plasmid standard DNAs, respectively. A 10-fold diluted DNA series of 105, 104, 103, 102, 10 copies per reaction were usedto assess sensitivity levels. PCR cycle conditions: 50℃ 2 min, 94℃ 2 min, 95℃ 15 s, 60℃ 45 s; 45 cycles. 11 pathogen DNAs ofActinomyces isrealii, Aerococcus viridians, Enterococcus faecalis, Enterobacter cloacae, Morganella morganii, Mycoplasma hominis,Pseudomonas aeruginosa, Streptococcus epidermidis, Candida albicans, Herpes simplex virus 1 and Herpes simplex virus 2 withurinary tract infection were selected to confirm the specificity.2. Results and Discussion2.1 Primer and probe screeningIn this study, the specificity of primers was screened by using human whole genome DNA and other pathogens’ nucleic acid asPCR template. Based on the results of specific amplification and sequencing, primer pairs of C1f/C1r; N1f/N1r; U5f/U5r were usedfor multiple PCR as shown in Figure 1. According to the screened primer sequence, the optimized primers and probes' sequences inTable 1 were finally selected for multiplex real-time PCR system.Volume 11 | Issue 2 - 13 -Figure 1Multiple PCR analysis of mixed 3 primer pairs. CT/NG/UU (lane 1), NG/UU (lane 2), CT/UU (lane 3), NG/CT (lane 4), UUDNA (lane 5), NG DNA (lane 6), CT DNA (lane 7).Table 1 Optimized primers and probes2.2 Multiplex real-time PCR assaySimultaneous amplification and detection of the three pathogens were achieved using the screened primers and fluorescentprobes listed in Table 1 and verified by electrophoresis (Figure 1). In the presence of a single target, the multiplex PCR produceddetection limits of 10 copies of CT, NG or UU plasmid control DNAs, respectively, whereas detection limits were unchanged whentwo targets (CT/NG, CT/UU, or UU/NG) or all three targets were present in the PCR mixture.Advanced Emergency Medicine- 14 -Figure 2 Evaluation of the limit of multiplex PCR. DNA copy number gradients for all three pathogens were 1 copy, 10 copies and100 copies per reaction.Probe-based multiplex real-time PCR could simultaneously distinguish multiple pathogens (Carrillo et al. 2020). Here, newprobes as shown in Table 1 were designed and individually examined with crude DNAs of CT, NG and UU (data not shown) by themultiplex real-time PCR. The developed multiplex real time PCR of high throughput was successfully established in this survey andcan be used as a simple and useful alternative to the monoplex real-time PCR for rapid and accurate identification of CT, NG and UU.3. AcknowledgementsWe thank Hang Zhou KingMed Diagnostics Company for providing the DNA samples of clinical specimens. This work wassupported by Zhejiang Provincial Natural Science Foundation of China (Grant No. LGF18C050002) and Zhejiang Medical and HealthScience and Technology Project (Grant No. 2018PY030 and 2020KY629).4. Conflict of interestThe authors declare that they have no conflicts of interest.5. References[1] Xu, WH., Chen, JJ., Sun, Q., Wang, LP., Jia, YF., Xuan, BB., Xu, B., & Sheng, HM.(2018). Chlamydia trachomatis, Ureaplasmaurealyticum and Neisseria gonorrhoeae among Chinese women with urinary tract infections in Shanghai: A community-basedcross-sectional study. J Obstet Gynaecol Res 44 (3), 495-502.[2] Roy, A., Dadwal, R., Yadav, R., Singh, P., Krishnamoorthi, S., Dasgupta, A., Chakraborti, A., & Sethi, S. (2021). Association ofChlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Ureaplasma species infection and organism load withcervicitis in north Indian population.Lett Appl Microbiol 73 (4), 506-514[3] Liang, YY., Zhai, HY., Li, ZJ., Jin, X., Chen, Y., & Chen, SP. (2018). Prevalence of Ureaplasma urealyticum, Chlamydiatrachomatis, Neisseria gonorrhoeae and herpes simplex virus in Beijing, China. Epidemiol Infect 147, e59.[4] Vallely, LM., Egli-Gany, D., Pomat, W., Homer, CS., Guy, R., Wand, H., Silver, B., Rumbold, AR., Kaldor, JM., Low, N., &Vallely, AJ. (2018). Adverse pregnancy and neonatal outcomes associated with Neisseria gonorrhoeae, Mycoplasma genitalium, M.hominis, Ureaplasma urealyticum and U. parvum: a systematic review and meta-analysis protocol. BMJ open 8 (11), e024175.[5] Horner, P., Donders, G., Cusini, M., Gomberg, M., Jensen, JS., & Unemo, M. (2018). Should we be testing for urogenitalMycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum in men and women? - a position statement from theVolume 11 | Issue 2 - 15 -European STI Guidelines Editorial Board. J Eur Acad Dermatol Venereol 32 (11), 1845–1851.[6] Bartoletti, R., Wagenlehner, F., Bjerklund Johansen, TE., Köves, B., Cai, T., Tandogdu, Z., & Bonkat, G. (2019). Management ofurethritis: Is it still the time for empirical antibiotic treatments?. Eur Urol Focus 5 (1), 29–35.[7] Marangoni, A., Foschi, C., Nardini, P., D'Antuono, A., Banzola, N., Di Francesco, A., & Cevenini, R. (2012). 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Simultaneous Detection of Chlamydia Trachomatis, Neisseria Gonorrhoeae, Ureaplasma Urealyticum by Multiplex PCR-Running

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Simultaneous Detection of Chlamydia Trachomatis, Neisseria Gonorrhoeae, Ureaplasma Urealyticum by Multiplex PCR-Running

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