Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions
Background: Sexually transmitted diseases (STD) are a major cause of infertility, long-term disability, ectopic pregnancy, and premature birth. Therefore, the development of fast and low-cost laboratory STD diagnostic screening methods will contribute to reducing STD-induced reproductive tract damag...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-11-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fcimb.2019.00382/full |
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| author | Xiao-Mei Hu Xiao-Mei Hu Jiang-Xia Xu Li-Xia Jiang Lian-Rui Deng Zhen-Mei Gu Xiao-Ying Xie Hui-Cai Ji Wei-Hua Wang Wei-Hua Wang Li-Ming Li Cheng-Nan Tian Fang-Li Song Shao Huang Lei Zheng Tian-Yu Zhong Tian-Yu Zhong |
| author_facet | Xiao-Mei Hu Xiao-Mei Hu Jiang-Xia Xu Li-Xia Jiang Lian-Rui Deng Zhen-Mei Gu Xiao-Ying Xie Hui-Cai Ji Wei-Hua Wang Wei-Hua Wang Li-Ming Li Cheng-Nan Tian Fang-Li Song Shao Huang Lei Zheng Tian-Yu Zhong Tian-Yu Zhong |
| author_sort | Xiao-Mei Hu |
| collection | DOAJ |
| description | Background: Sexually transmitted diseases (STD) are a major cause of infertility, long-term disability, ectopic pregnancy, and premature birth. Therefore, the development of fast and low-cost laboratory STD diagnostic screening methods will contribute to reducing STD-induced reproductive tract damage and improve women's health worldwide. In this study, we evaluated a novel multiplex real-time PCR melting curve assay method for the simultaneous detection of 9 STD pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, and herpes simplex virus.Methods: The analytical performance of the method, including its limit of detection (LOD), specificity, repeatability, and effect on different DNA extraction kits were evaluated. Additionally, we obtained 1,328 clinical specimens from 3 hospitals to detect the 9 STD pathogens using multiplex real-time PCR melting curve and Sanger sequencing, to evaluate the sensitivity, specificity, and consistency of the assay method.Results: The results showed that the analytical sensitivity of the novel multiplex real-time PCR melting curve assay is very excellent, with LOD of DNA corresponding to <200 copies/μL for the DNA of the 9 STDs and 1.00 × 104 color change unit /ml for those of UU and UP. Additionally, this assay demonstrated excellent analytical specificity, excellent repeatability, and its results had no effect of different DNA extraction kits. The performance, in terms of sensitivity (91.06–100%) and specificity (99.14–100%), was remarkable, since the consistency between it and Sanger sequencing was more than 0.85 in the clinic.Conclusion: The novel multiplex real-time PCR melting curve assay method has high sensitivity and specificity, relatively low cost, and simple to use for the simultaneous detection of 9 STD pathogens in genitourinary secretions. |
| first_indexed | 2024-12-11T19:03:04Z |
| format | Article |
| id | doaj.art-4a2624d0285e48f2b071b4a5479ff125 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-12-11T19:03:04Z |
| publishDate | 2019-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-4a2624d0285e48f2b071b4a5479ff1252022-12-22T00:53:57ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882019-11-01910.3389/fcimb.2019.00382470858Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary SecretionsXiao-Mei Hu0Xiao-Mei Hu1Jiang-Xia Xu2Li-Xia Jiang3Lian-Rui Deng4Zhen-Mei Gu5Xiao-Ying Xie6Hui-Cai Ji7Wei-Hua Wang8Wei-Hua Wang9Li-Ming Li10Cheng-Nan Tian11Fang-Li Song12Shao Huang13Lei Zheng14Tian-Yu Zhong15Tian-Yu Zhong16Department of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Precision Medicine Center, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Medical Laboratory, The Fourth Affiliated Hospital of Nanchang University, Nanchang, ChinaDepartment of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Medical Laboratory, The Fourth Affiliated Hospital of Nanchang University, Nanchang, ChinaDepartment of Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaDepartment of Obstetrics and Gynecology, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaDepartment of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Precision Medicine Center, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Dermatology, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Cardiac and Thoracic Surgery, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaJiangxi Shiningmed Medical Technology Ltd., Ganzhou, ChinaJiangxi Shiningmed Medical Technology Ltd., Ganzhou, ChinaDepartment of Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaDepartment of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaDepartment of Precision Medicine Center, First Affiliated Hospital of Gannan Medical University, Ganzhou, ChinaBackground: Sexually transmitted diseases (STD) are a major cause of infertility, long-term disability, ectopic pregnancy, and premature birth. Therefore, the development of fast and low-cost laboratory STD diagnostic screening methods will contribute to reducing STD-induced reproductive tract damage and improve women's health worldwide. In this study, we evaluated a novel multiplex real-time PCR melting curve assay method for the simultaneous detection of 9 STD pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, and herpes simplex virus.Methods: The analytical performance of the method, including its limit of detection (LOD), specificity, repeatability, and effect on different DNA extraction kits were evaluated. Additionally, we obtained 1,328 clinical specimens from 3 hospitals to detect the 9 STD pathogens using multiplex real-time PCR melting curve and Sanger sequencing, to evaluate the sensitivity, specificity, and consistency of the assay method.Results: The results showed that the analytical sensitivity of the novel multiplex real-time PCR melting curve assay is very excellent, with LOD of DNA corresponding to <200 copies/μL for the DNA of the 9 STDs and 1.00 × 104 color change unit /ml for those of UU and UP. Additionally, this assay demonstrated excellent analytical specificity, excellent repeatability, and its results had no effect of different DNA extraction kits. The performance, in terms of sensitivity (91.06–100%) and specificity (99.14–100%), was remarkable, since the consistency between it and Sanger sequencing was more than 0.85 in the clinic.Conclusion: The novel multiplex real-time PCR melting curve assay method has high sensitivity and specificity, relatively low cost, and simple to use for the simultaneous detection of 9 STD pathogens in genitourinary secretions.https://www.frontiersin.org/article/10.3389/fcimb.2019.00382/fullmultiplexpolymerase chain reactionsanger sequencingsexually transmitted diseasespathogen |
| spellingShingle | Xiao-Mei Hu Xiao-Mei Hu Jiang-Xia Xu Li-Xia Jiang Lian-Rui Deng Zhen-Mei Gu Xiao-Ying Xie Hui-Cai Ji Wei-Hua Wang Wei-Hua Wang Li-Ming Li Cheng-Nan Tian Fang-Li Song Shao Huang Lei Zheng Tian-Yu Zhong Tian-Yu Zhong Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions Frontiers in Cellular and Infection Microbiology multiplex polymerase chain reaction sanger sequencing sexually transmitted diseases pathogen |
| title | Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions |
| title_full | Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions |
| title_fullStr | Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions |
| title_full_unstemmed | Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions |
| title_short | Design and Evaluation of a Novel Multiplex Real-Time PCR Melting Curve Assay for the Simultaneous Detection of Nine Sexually Transmitted Disease Pathogens in Genitourinary Secretions |
| title_sort | design and evaluation of a novel multiplex real time pcr melting curve assay for the simultaneous detection of nine sexually transmitted disease pathogens in genitourinary secretions |
| topic | multiplex polymerase chain reaction sanger sequencing sexually transmitted diseases pathogen |
| url | https://www.frontiersin.org/article/10.3389/fcimb.2019.00382/full |
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