A rapid method for detecting prfA and hly toxin genes of Listeria monocytogenes using double nucleic acid colloidal gold strips

doi:10.12122/j.issn.1673-4254.2025.02.20

Abstract

Abstract:

Objective To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. Methods L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. Results The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. Conclusion The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.

Key words: Listeria monocytogenes, prfA, hly, double nucleic acid test strip

Yan LIU, Jianyu YANG, Yujiao ZHOU, Wenbo DING, Xianyu ZHANG, Linran GAO, Beizhen PAN, Jifei YANG, Yundong ZHAO. A rapid method for detecting prfA and hly toxin genes of Listeria monocytogenes using double nucleic acid colloidal gold strips[J]. Journal of Southern Medical University, 2025, 45(2): 387-394.

Figures/Tables 17

Tab.1 Specific primers for PCR amplification of Listeria monocytogenesprfA and hly genes

Specific primer	Specific primer sequence (5'-3')
prfA	F:(6-FAM)-GCTGATTACCGAGAAGGGAA
prfA	R:(Biotin)-CCGCTAACGAGTGGATAAGA
hly	F:(Digoxin)-TATACCACGGAGATGCAGTG
hly	R:(Biotin)-AGCACCTGGATATGTTAGGC

Tab.2 Composition of the double toxin detection kit

Constituent	Quantity/Unit	Effect	Storage mode
ddH₂O	4 mL	DNA extraction and blank control	-20 ℃
PCR tube	20	PCR amplification	-20 ℃
Positive standard solution	20 μL	Positive control	-20 ℃
Negative standard solution	20 μL	Negative control	-20 ℃
Colloidal gold test strip	20	Visualization	-20 ℃

Fig.1 Electrophoresis of the cloned target gene fragments. A: Blue and white spot screening. B: Electrophoresis of prfA and hly plasmids. M: 100bp Marker; 1: Plasmid prfA; 2: Plasmid hly; N: Blank control.

Fig.2 Comparison results of prfA gene sequencing in Listeria monocytogenes.

Fig.3 Sequencing results of Listeria monocytogenes hly gene.

Fig.4 Specificity assessment of the double virulence gene detection kit. 1: Listeria monocytogenes; 2: Staphylococcus aureus; 3: Escherichia coli; 4: Bacillus cereus; N: Blank control.

Fig.5 Specificity assessment of the kit with 1.5% agarose gel electrophoresis. M:100 bp Marker; 1: Listeria monocytogenes; 2: Staphylococcus aureus; 3: Escherichia coli; 4: Bacillus cereus; N: Blank control.

Fig.6 Electrophoretic sensitivity assessment of the dual virulence gene detection kit. M: 100 bp Marker; 1: 101 ng/μL; 2: 100 ng/μL; 3: 10-1 ng/μL; 4: 10-2 ng/μL; 5: 10-3 ng/μL; 6: 10-4 ng/μL; N: Blank control.

Fig.7 Sensitivity assessment of the kit with 1.5% agarose gel electrophoresis. M:100 bp Marker; 1: 101 ng/μL; 2: 100 ng/μL; 3: 10-1 ng/μL; 4: 10-2 ng/μL; 5: 10-3 ng/μL; 6: 10-4 ng/μL; N: Blank control.

Fig.8 Assessment of detection result reproducibility of the double virulence gene detection kit. 1-3: Listeria monocytogenes; N: Blank control.

Fig.9 Assessment of reproducibility of the kit using 1.5% agarose gel electrophoresis. M:100 bp Marker;1-3: Listeria monocytogenes; N: Blank control.

Fig.10 Stability test results of the double virulence gene test kit. A: 6th month. B: 9th month. C: 12th month. 1, 3, 5: Listeria monocytogenes; 2, 4, 6: Blank control.

Fig.11 Stability test of the kit using 1.5% agarose gel electrophoresis. A: 6th month. B: 9th month. C: 12 th month. M: 100 bp Marker;1:Listeria monocytogenes; N: Blank control.

Fig.12 Results of dual virulence gene detection kit for contaminated samples. 1: 1.5×108 CFU/mL; 2: 1.5×107 CFU/mL; 3: 1.5×106 CFU/mL; 4: 1.5×105 CFU/mL; 5: 1.5×104 CFU/mL; 6: 1.5×103 CFU/mL; 7: 1.5×102 CFU/mL; 8: 1.5×101 CFU/mL; N: Blank control.

Fig.13 1.5% agarose gel electrophoresis for verification of the detection results using artificially contaminated samples. M:100 bp Marker; 1: 1.5×108 CFU/mL; 2: 1.5×107 CFU/mL; 3: 1.5×106 CFU/mL; 4: 1.5×105 CFU/mL; 5: 1.5×104 CFU/mL; 6: 1.5×103 CFU/mL; 7: 1.5×102 CFU/mL; 8: 1.5×101 CFU/mL; N: Blank control.

Fig.14 Detection results of actual samples using double virulence gene test kit. 1: Positive control; 2: Raw milk samples1; 3: Raw milk 2; 4: Raw milk 3; 5: Raw milk 4; 6: Raw beef; 7: Cooked meat product; 8: Quick-frozen aquatic products; 9: Vegetable salad; 10: Frozen drink; 11: Watermelon; 12: Blank control.

Fig.15 1.5% agarose gel electrophoresis for verification of the test results of using actual samples. M: 100 bp Marker; 1: Positive control; 2: Raw milk samples1; 3: Raw milk 2; 4: Raw milk 3; 5: Raw milk 4; 6: Raw beef; 7: Cooked meat product; 8: Quick-frozen aquatic products; 9: Vegetable salad; 10: Frozen drink; 11: Watermelon; N: Blank control.

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