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Development of multiplex mpn pcr to detect major vibrio species in raw fish
- 1. DEVELOPMENT OF MULTIPLEX MPN-PCR TO DETECT MAJOR VIBRIO SPECIES IN RAW FISH Sharmin Quazi Bonny
- 2. Fish & fish products are the 2nd most important Protein source. BBC: Global fish consumption per capita has made a record in 2016 .
- 3. • Vibrio is a gram negative, rod- shaped bacteria • Responsible for an estimated 80,000 illnesses and 100 deaths in the United States every year. V. parahaemolyticus V. cholerae V. vulnificus
- 4. Advantage and Limitations of Current Methods Conventional Method MPN (Most Probable Number) Method PCR Multiplex PCR Simplex MPN- PCR Reverse Transcriptase- qPCR • Generally recognized as the classical method (Biochemical and serological tests). • Sensitive • Can quantify • Can detect the presence of Live bacteria • Fast and robust method • Reduced cost and time since many species can be detected in a single assay • Capable of quantifying • Can specify the presence of live bacteria • Highly Sensitive • Capable of quantifying • Can detect the live bacteria • Laborious, time- consuming, and complex process. • The specificity of using selective media is still questionable. • Laborious, time- consuming, and complex process. • The specificity of using selective media is still questionable. • Cannot quantify • Cannot differentiate between the presence of live and dead bacteria • Cannot quantify • Cannot differentiate between the presence of live or dead bacteria • Cannot detect more than one species in a single assay platform • RNA is less stable in compare to DNA • Cannot be used in small-scale laboratory
- 5. The developed Method MPN Assay • Sensitive • Can Quantify • Can detect the presence of life bacteria Multiplex MPN-PCR • Highly Sensitive • Capable of Quantifying • Can specify the presence of live bacteria • Capable of detecting more than one species in a single platform Multiplex PCR • Reduce cost and time in compare t Simplex PCR since many species can be detected in single assay
- 6. Primers Used Target Bacteria Target Gene Amplicon Size Primers Forward Primer Reverse Primer Vibrio parahaemolyticus pntA 409 5’-AGCAAGTTTCGATGATGCTG-3’ 5’-ACCAGCAACCAAAACTTTCGCT-3’ Vibrio Cholerae pntA 338 5’-CAGTAAAGAAACGACCAAACTC-3’ 5’-TGCCAGTTTCGATGATGCCG-3’ Vibrio vulnificus vvhA 205 5′-TTCCAACTTCAAACCGAACTATGAC-3′ 5′-ATTCCAGTCGATGCGAATACGTTG-3′
- 7. Work Flow IAC (Internal Amplification Control) was used in PCR DNA was extracted using boil cell method Multiplex PCR were optimize Reference strains of V. parahaemolyticus, V. cholerae & V. vulnificus were used Primers targeting pnt A gene for both V. parahaemolyticus and V. cholerae and vvh A gene for V. vulnificus were used
- 8. 1 ml of food homogenate (1.5 ml Microcentrifuge Tube) Discard Supernatant Centrifuge at 12,000 rpm for 3 min Re-suspend the Pellet in 100 µl of 1X PBS buffer, pH: 7.4 Centrifuge at 12,000 rpm for 2 min Discard Supernatant Re-suspend the Pellet in 100 µl of sterile distilled water Boiled for 5 min Snapped cooled in ice for 10 min Centrifuge at 12,000 rpm for 3 min Collect Supernatant (80 µl) Stored at -20°C until further use Repeatedtwice DNA Extraction Method (Boil Cell)
- 9. Raw Fish (N=120) Pre-enrichment (37℃ for 6 h) 7 times 10-fold dilution in SPB* Enrichment (37℃ for 14-16h) DNA Extraction MPN Analysis Multiplex PCR Tryptic Soy Broth (TSB) + 3% Nacl Simplex PCR DNA Extraction Multiplex PCR
- 10. Results
- 11. Work Flow 50 bp 200 bp 150 bp 100 bp 400 bp 300 bp 250 bp 650 bp 800 bp 500 bp V. Cholerae (338 bp) V. Vulnificus (205 bp). IAC (187 bp) V. Parahaemolyticus (409 bp) Multiplex-PCR for V. parahaemolyticus (409 bp) ,V.cholerae (338 bp), and V. vulnificus (205 bp). The pntA gene was targeted for V.cholerae and V. parahaemolyticus, whereas vvhA gene was targeted for V. vulnificus. Shown are: Lane 1: Negative control; Lane 2: IAC (Internal Amplification Control); Lane 3: V.vulnificus & IAC; Lane 4: V. cholerae & IAC; Lane 5: V. parahaemolyticus & IAC; Lane 6: V. cholera , V. parahaemolyticus & IAC. Lane 7: Optimized tetraplex PCR with three vibrio and IAC; Lane 8: 50-bp DNA ladder. 1 52 43 876
- 12. 34.16% 18.33% 22.50% 48.33% 27% 32.50% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% V. parahaemolyticus V. cholerae V. vulnificus PERCENTAGE Multiplex PCR Vs. Multiplex MPN-PCR Multiplex PCR Multiplex MPN-PCR To assess the Difference in Sensitivity Comparison of the presence of three major Vibrios in raw fish using Multiplex PCR Vs. Multiplex MPN-PCR
- 13. Comparison of the presence of three major Vibrios in raw fish using Multiplex PCR Vs. Multiplex MPN-PCR 34.16% 18.33% 22.50% 48.33% 27% 32.50% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% V. parahaemolyticus V. cholerae V. vulnificus PERCENTAGE Multiplex PCR Vs. Multiplex MPN-PCR Multiplex PCR Multiplex MPN-PCR
- 14. Sample Number Vibrio Parahaemolyticus Vibrio cholerae Vibrio vulnificus Simplex PCR Multiplex PCR Simplex PCR Multiplex PCR Simplex PCR Multiplex PCR PCR Positive % PCR Positive % PCR Positive % PCR Positive % PCR Positive % PCR Positive % 120 58 48 58 48 32 27 32 27 39 32 39 32 Comparison of the enumeration data of the Simplex & Multiplex MPN-PCR To validate the Multiplex MPN-PCR in terms of Simplex MPN-PCR method
- 15. Sample Number Vibrio Parahaemolyticus Vibrio cholerae Vibrio vulnificus Simplex PCR Multiplex PCR Simplex PCR Multiplex PCR Simplex PCR Multiplex PCR PCR Positive % PCR Positive % PCR Positive % PCR Positive % PCR Positive % PCR Positive % 120 58 48 58 48 32 27 32 27 39 32 39 32 Comparison of the enumeration data of the Simplex & Multiplex MPN-PCR
- 16. Distribution of MPN-PCR values of Vibrios in Raw Fish 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 ≤3 >3 to ≤1000 >1000 to ≤ 5000 >5000 Percentage MPN-PCR Value (MPN/g) VPS (%) VPM(%) VCS(%) VCM(%) VVS(%) VVM(%)
- 17. Relative abundance of two or more bacteria 10.83333333 14.166666671.666666667 6.666666667 0 5 10 15 VP-VC VP-VV VC-VV VP-VC-VV Total Sample: 120 Raw Fish
- 18. Conclusion A multiplex MPN-PCR method was optimized and validated for screening of three most virulent Vibrios in the laboratory. Incorporation of MPN techniques with multiplex PCR assay is an advantage because it allows quantitative detection of multiple species in a single assay platform. The occurrence of Vibrios in raw fish was determined by using three methods including multiplex PCR, simplex MPN-PCR and multiplex MPN- PCR. V. parahaemolyticus, V. cholera and V. vulnificus were observed in 43.33%, 27%, and 32.5% raw fish samples, respectively with both simplex and multiplex MPN-PCR assay. MPN-PCR could detect 9.17%, 8.67% and 10% more of V. parahaemolyticus, V. cholera and V. vulnificus, respectively in the food samples as compared to the lower percentages using the conventional multiplex PCR.