Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection

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Microbiology

Main article text

 

Introduction

Materials and Methods

Bacterial strains

Clinical stool specimens

Informed consent statement

DNA extraction

Primer design

LAMP reaction

Detection of LAMP products

PCR detection

Statistical analysis

Results

Optimal primers for rapid detection of tcdA

Appropriate temperature for tcdA LAMP reaction

Specificity of tcdA LAMP reaction

Sensitivity of tcdA LAMP reaction

Evaluation of the assay with stool specimens

Discussion

Conclusions

Supplemental Information

Sequence comparison of tcdA gene carried by Clostridioides difficile VPI10463 with the reported tcdA gene in GenBank.

DOI: 10.7717/peerj.17776/supp-1

STROBE checklist.

DOI: 10.7717/peerj.17776/supp-2

Additional Information and Declarations

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Minyi Lin conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Pu Wang conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, funding Acquisition, and approved the final draft.

Bingyun Lu conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Ming Jin conceived and designed the experiments, performed the experiments, prepared figures and/or tables, and approved the final draft.

Jiasheng Tan conceived and designed the experiments, performed the experiments, prepared figures and/or tables, and approved the final draft.

Wei Liu conceived and designed the experiments, performed the experiments, prepared figures and/or tables, technical support, and approved the final draft.

Jing Yuan conceived and designed the experiments, performed the experiments, prepared figures and/or tables, technical support, and approved the final draft.

Xiaomou Peng conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, project Administration, and approved the final draft.

Ye Chen conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, funding Acquisition; Project Administration, and approved the final draft.

Human Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

Fudan University Affiliated Huashan Hospital granted Ethical approval to carry out the study within its facilities (Ethical Application Ref: FDEC-2012-014).

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

The tcdA gene sequences of C. difficile are available at GenBank: X92982.1.

https://www.ncbi.nlm.nih.gov/nuccore/X92982.1/

Data Availability

The following information was supplied regarding data availability:

The sequence comparison of tcdA gene carried by Clostridioides difficile VPI10463 with the reported tcdA gene in GenBank are available in the Supplemental Files. The raw data includes Figs. 1, 2, 3A, 3B, 4A4C, and Table 3.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 82270581), the GuangDong Basic and Applied Basic Research Foundation (grant number 2022A1515220213), the National Key Research and Development Program of China (grant number 2021YFA0717001), and the Shenzhen Science and Technology Plan Project (grant number KCXFZ20211020163558024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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