Rapid and high-throughput identification of recombinant bacteria with mass spectrometry assay.
OBJECTIVE
To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry.


METHODS
Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques were used to identify 12 recombinant proteins (10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori). A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-ClinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FlexAnalysis softwares.


RESULTS
Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities: RC=100%, mean CVA=98.7% (the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively) and PPV=95%. This model can be used to classify the bacteria and their recombinant, which only requires 3.7×103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks (9, 16, 9, and 14 for the four stages, respectively) was found in the various phase of recombinant bacteria.


CONCLUSIONS
MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China.
Di
D
Xiao
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China.
Xiao Xia
XX
Tao
Provincial Key Laboratory for Plague Control and Prevention, Yunnan Provincial Institute for Endemic Diseases Control and Prevention, Dali 671000, Yunnan, China.
Peng
P
Wang
Beijing Municipal Center for Disease Control and Prevention, Beijing 102206, China.
Guo Dong
GD
Liu
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China.
Ya Nan
YN
Gong
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China.
Hui Fang
HF
Zhang
Chaoyang District Center for Disease Control and Prevention, Beijing 100021, China.
Hai Bin
HB
Wang
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China.
Jian Zhong
JZ
Zhang
D001426:Bacterial Proteins / Q000032:analysis*; D003001:Cloning, Molecular; D004926:Escherichia coli; D013058:Mass Spectrometry; D030781:Organisms, Genetically Modified; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis*
D001426:Bacterial Proteins; D011994:Recombinant Proteins
ClinProTools; MALDI-TOF MS; Rapid identification; Recombinant bacteria; Specific peaks