Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood
ABSTRACT PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA ex...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-06-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.05198-22 |
| _version_ | 1797803404108496896 |
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| author | Zi-Han Wei Lin Xie Yong-Jing Wang Jiang-Xing Zhuang Jian-Jun Niu Li-Li Liu |
| author_facet | Zi-Han Wei Lin Xie Yong-Jing Wang Jiang-Xing Zhuang Jian-Jun Niu Li-Li Liu |
| author_sort | Zi-Han Wei |
| collection | DOAJ |
| description | ABSTRACT PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 106 to 100 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 102 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA. |
| first_indexed | 2024-03-13T05:20:15Z |
| format | Article |
| id | doaj.art-8bfdc8957bf940a0ae9449318a4d2a0a |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-13T05:20:15Z |
| publishDate | 2023-06-01 |
| publisher | American Society for Microbiology |
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| series | Microbiology Spectrum |
| spelling | doaj.art-8bfdc8957bf940a0ae9449318a4d2a0a2023-06-15T13:18:33ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-06-0111310.1128/spectrum.05198-22Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from BloodZi-Han Wei0Lin Xie1Yong-Jing Wang2Jiang-Xing Zhuang3Jian-Jun Niu4Li-Li Liu5Center of Clinical Laboratory, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, ChinaCenter of Clinical Laboratory, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, ChinaCenter of Clinical Laboratory, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, ChinaFujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research, Institute of Neuroscience, School of Medicine, Xiamen University, Xiamen, ChinaCenter of Clinical Laboratory, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, ChinaCenter of Clinical Laboratory, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, ChinaABSTRACT PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 106 to 100 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 102 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA.https://journals.asm.org/doi/10.1128/spectrum.05198-22PCRsample preparationbloodblood componentDNA yieldTreponema pallidum |
| spellingShingle | Zi-Han Wei Lin Xie Yong-Jing Wang Jiang-Xing Zhuang Jian-Jun Niu Li-Li Liu Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood Microbiology Spectrum PCR sample preparation blood blood component DNA yield Treponema pallidum |
| title | Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood |
| title_full | Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood |
| title_fullStr | Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood |
| title_full_unstemmed | Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood |
| title_short | Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood |
| title_sort | red blood cell lysis pretreatment can significantly improve the yield of treponema pallidum dna from blood |
| topic | PCR sample preparation blood blood component DNA yield Treponema pallidum |
| url | https://journals.asm.org/doi/10.1128/spectrum.05198-22 |
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