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An improved method on isolation and serial passage of Chlamydia pneumoniae from human peripheral blood mononuclear
Qian Jin1, Feihua Huang, Shuming Sun
1Department of Respiratory Medicine, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang, China.
Background:
Conventional method for Chlamydia pneumoniae (Cpn) isolation and propagation is technically challenging and time-consuming. Here, we developed a method to improve the isolation and passage of Cpn collected from human peripheral blood mononuclear cells (PBMCs).
Methods:
PBMCs positive with Cpn antigen (Cpn-Ag) were isolated, then centrifuged and cultured with Hep-2 cells after being broken. Cells were broken again and put into new Hep-2 cells to finish totally four passages with isolated and imported Cpn. Microimmunofluorescence method was used to detect Cpn. Inclusion forming unit (IFU) number was counted for each passage. Polymerase chain reaction (PCR) method was used to detect Cpn DNA. Efficiency of different centrifugation modes was compared.
Results:
Hep-2 cells of the first and second passages were strong positive with Cpn-Ag, the third passage was positive, and the fourth negative. Degeneration appeared in the fourth passage for isolated Cpn and third passage for imported strain. Centrifugation mode of 1,000 rpm for 2 h was the most efficient for Cpn propagation and passage.
Conclusion:
This simplified method achieved efficient isolation, propagation, and passage of Cpn from PBMCs, and isolated strain was superior to imported strain on propagating ability.
Insights
A new, simplified method efficiently isolates and propagates Chlamydia pneumoniae (Cpn) from human peripheral blood mononuclear cells (PBMCs). This technique improves Cpn isolation and passage, outperforming conventional methods.
Area of Science:
- Microbiology
- Infectious Diseases
- Cell Biology
Background:
- Conventional Chlamydia pneumoniae (Cpn) isolation and propagation are difficult and slow.
- A novel method was developed to enhance Cpn isolation and passage from human peripheral blood mononuclear cells (PBMCs).
Purpose of the Study:
- To develop and validate an improved method for isolating and propagating Chlamydia pneumoniae from human PBMCs.
- To assess the efficiency of Cpn isolation and passage using the developed method.
Main Methods:
- Human PBMCs positive for Cpn antigen were isolated, lysed, and co-cultured with Hep-2 cells for propagation.
- Multiple passages (up to four) were performed, with Cpn detection using micro-immunofluorescence and PCR.
- The efficiency of different centrifugation speeds and durations was evaluated for optimal Cpn propagation.
Main Results:
- The simplified method demonstrated successful isolation and propagation of Cpn through multiple passages.
- Hep-2 cells showed strong positive Cpn antigen results in the first two passages, with positive results in the third and negative in the fourth.
- Optimal Cpn propagation and passage were achieved using centrifugation at 1,000 rpm for 2 hours.
Conclusions:
- The developed simplified method enables efficient isolation, propagation, and passage of Chlamydia pneumoniae from PBMCs.
- The Cpn strain isolated using this method exhibited superior propagating ability compared to an imported strain.
