Establishment of reference material of multiplex nucleic acid assay for bacterial infection

LIU Dong-lai; ZHOU Hai-wei; SHI Da-wei; SHEN Shu; TIAN Ya-bin; ZHANG Chun-tao

doi:10.3969/j.issn.1007-8134.2018.03.004

Abstract：Objective To establish reference material of multiplex nucleic acid assays for identification of bacterial infection and formulate relevant quality standards, thus providing evidence for quality assessment of related products. Methods Thirty-four pathogenic bacteria and fungi associated with central nervous system, blood, respiratory, intestinal and reproductive tract infections were selected for further culture and enrichment, and were identified by using fully automated microbial identification system and drug sensitive analyzer VITEK2. The gradient dilution method was used to determine the concentration of identified correct strains, then all strains at a high concentration were grouped and mixed as reference material candidates. The composition and specificity of mixed candidates were verified by using the whole genome sequencing. Collaboration study was conducted using multiplex nucleic acid assays based on different detection principles to determine the quality standards of reference material. Finally, the stability of the reference material was evaluated. Results Total 34 pathogenic bacteria and fungi, including 28 bacteria and 6 fungi, were mixed as 7 reference materials coded M1—M7 (final concentration of bacteria was 1×108—1×109CFU/ml and final concentration of fungi was 1×106—1×107CFU/ml). The whole genome second-generation sequencing technology was applied to verify that the reference product was composed correctly and there was no cross-contamination. Reference product was tested with multiplex nucleic acid assays based on PCR-melting curves, isothermal amplification and targeted-amplification second-generation sequencing technologies. The results showed that detecting the suitably diluted reference material could be used to evaluate the multiplex nucleic acid assays, such as accuracy, specificity, reproducibility and limit of detection, and there was no cross-containment among mixed strains. The stability study showed that 3 times freeze-thawing cycles did not impact the stability of reference material. Conclusions The reference material and its quality standards have been established and it can be used for quality evaluation of multiplex nucleic acid assays for identification of bacterial infection.