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China Biotechnology
China Biotechnology  2020, Vol. 40 Issue (4): 34-41    DOI: 10.13523/j.cb.1910009
Accurate Detection of Streptococcus pneumoniae by Using Ribosomal Protein L7 / L12 as Molecular Marker
WANG Meng1,SONG Hui-ru1,CHENG Yu-jie1,WANG Yi1,2,3,YANG Bo1,2,3,HU Zheng1,2,3,**()
1 School of Food and Biological Engineering, Hubei University of Technology, Wuhan 430068, China
2 Key Laboratory of Fermentation Engineering (Hubei University of Technology), Ministry of Education, Wuhan 430068, China
3 Hubei Collaborative Innovation Center for Industrial Fermentation, Wuhan 430068, China
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Objective: A rapid colloidal gold immunochromatography assay (GICA) for the detection of Streptococcus pneumoniae was explored by using the ribosomal protein L7/L12 as detection marker. Method: The gene sequence of ribosomal protein L7/L12 was analyzed, the prokaryotic expression vector was constructed, the recombinant protein was expressed and purified, and the monoclonal antibody was prepared by immunizing BALB/c mice; The affinity of antigens and antibodies was detected by a labelless molecular interaction instrument based on BLI technology; The colloidal gold immunochromatographic test strip was developed based on the principle of double antibody sandwich, and its specificity, sensitivity and stability were evaluated. Result: Two hybridoma cell strains that can efficiently secrete anti-monoclonal antibody were acquired by means of screening. Purified monoclonal antibodies all had high affinity and no competition with antigen. Pairing and detection were made on colloidal gold immunochromatography platform. The minimum detection limit of the test strips was 1.0×105 CFU/ml; It had a specific reaction with Streptococcus pneumoniae, but did not cross-react with other 9 kinds of common respiratory pathogens such as Haemophilus influenzae and Moraxella catarrhalis; Test strips were kept at 25°C for 12 months and still had good repeatability and stability. Conclusions: RP-L7/L12 can be used as a detection marker for Streptococcus pneumonia. The colloidal gold immunochromatographic test strips prepared by its monoclonal antibody can be used for rapid detection of streptococcus pneumoniae.

Key wordsStreptococcus pneumoniae      Ribosomal protein L7/L12      Monoclonal antibody      Colloidal gold immunochromatography     
Received: 10 October 2019      Published: 18 May 2020
ZTFLH:  Q819  
Corresponding Authors: Zheng HU     E-mail:
Cite this article:

WANG Meng,SONG Hui-ru,CHENG Yu-jie,WANG Yi,YANG Bo,HU Zheng. Accurate Detection of Streptococcus pneumoniae by Using Ribosomal Protein L7 / L12 as Molecular Marker. China Biotechnology, 2020, 40(4): 34-41.

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Primer sequences Restriction enzyme cutting site
Table 1 Primer sequence of PCR amplified
Fig.1 Expression and purification of Sp-RP-L7/L12 M: protein marker; 1: pET-21a; 2: Uninduced negative control; 3: Total protein after induction of expression; 4: supernatant collected from ultrasound disrupted E. coli.; 5: precipitation collected from ultrasound disrupted E. coli.; 6: penetrating fluid; 7: eluent with 20 mmol/L imidazole; 8: eluent with 40 mmol/L imidazole; 9: eluent with 100 mmol/L imidazole; 10: eluent with 150 mmol/L imidazole
Fig.2 Identification and analysis of antibody purification by SDS-PAGE M: protein marker; 1: Sp-1#; 2: Sp-2#
Fig.3 Determination of antibody titer by indirect ELISA (a) Sp-1# antibody titer (b) Sp-2# antibody titer.
Subtype Lp-1 Lp-2
H-chain IgG1 1.043 2 1.429
IgG2a 0.249 9 0.214 5
IgG2b 0.145 2 0.204 8
IgG3 0.068 2 0.063 2
IgA 0.13 0.088 4
IgM 0.097 4 0.091 5
L-chain Kappa 0.500 3 0.616
Lambda 0.086 4 0.064 7
Table 2 Subtype identification of monoclonal antibodies
Fig.4 Identification of monoclonal antibodies SP-1# and SP-2# by Western blotting M: protein marker;1&3: recipitation of S. pneumoniae;2&4: recombinant protein RP-L7/L12
Fig.5 Detection of antibody affinity by molecular interaction instrument (a) Sp-1# binding and dissociation curves (b) Sp-2# binding and dissociation curves
Conc. (nM) Response KD (M) kon(1/Ms) kdis(1/s) Full R2
SP-1# 1 000 4.876 1 9.05E-09 2.64E+04 2.39E-04 0.997 1
500 3.897 7 8.86E-09 3.25E+04 2.88E-04 0.997 9
250 2.819 6 8.12E-09 4.38E+04 3.56E-04 0.998 3
125 2.442 8 9.29E-09 4.68E+04 4.35E-04 0.993 2
62.5 1.546 5 5.91E-09 6.92E+04 4.09E-04 0.998 1
SP-2# 1 000 3.887 7 3.67E-09 5.96E+04 2.19E-04 0.995 1
500 2.806 0 5.36E-09 7.89E+04 4.23E-04 0.992 8
250 1.900 2 6.51E-09 8.89E+04 5.79E-04 0.992 5
125 1.410 1 7.72E-09 4.05E+04 3.13E-04 0.993 1
62.5 0.698 3 4.57E-09 9.36E+04 4.28E-04 0.991 2
Table 3 Data results of Biofilm Interference Technology (BLI) test
Fig.6 Verification of Sp antibody recognizing different epitopes by in-tandem assay
Fig.7 Specificity test of S. pneumoniae colloidal gold immunochromatography strip
Fig.8 Sensitive test of S. pneumoniae colloidal gold immunochromatography strip 1-6: represents the concentration of S. pneumoniae: 1.0×109CFU/ml, 1.0×108CFU/ml, 1.0×107CFU/ml, 1.0×106CFU/ml, 1.0×105CFU/ml, 1.0×104CFU/ml; 7: negative control
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