Multiplex PCR detection primers, kits, methods and applications for identifying persistent Listeria monocytogenes st121
A mononuclear cell proliferation and detection kit technology, applied in the biological field, can solve problems such as reducing the throughput of multiplex PCR detection, and achieve the effects of saving reagent usage, improving detection efficiency, and simple and quick steps.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0033] Example 1 Single multiplex PCR system specific detection
[0034] The multiplex PCR kit for detecting the ST121 strain of Listeria monocytogenes, including conventional multiplex PCR components, also includes specific primers for the ST121 strain of Listeria monocytogenes, the specific primer sequences are shown in Table 1, routine Multiplex PCR kit including Taq DNA polymerase, MgCl 2 , PCR amplification buffer, dNTPs and deionized water.
[0035] Table 1 is aimed at the specific primer of Listeria monocytogenes ST121 strain
[0036]
[0037] Specific detection of single-plex PCR system: PCR amplification method of single primer pair + single template is used to amplify each target DNA sequence of Listeria monocytogenes ST121 strain. The templates of 0609, 0171 and prfA are all used For the genomic DNA of Listeria cellulogenes ST121 strain, the template DNA concentration is 20 ng / μL. Single-plex PCR adopts 25 μL reaction system, including: upstream and downstream...
Embodiment 2
[0038] The specific detection of embodiment 2 multiplex PCR system
[0039] 1. Specific detection of multiplex PCR system: multiple target DNA sequences of ST121 strain (Listeria monocytogenes ST121 strain) were amplified using multiple primer pairs + single template PCR amplification method, 0609, 0171, prfA Genomic DNA of the ST121 strain was used as the template, and the template DNA concentration was 20 ng / μL. Multiplex PCR uses 25 μL reaction system, including: 0.5 μL each of multiplex PCR primers 0609-F, 0609-R, 0171-F, 0171-R, prfA-F and prfA-R at a concentration of 10 μmol / L, 2.5 U / μL Taq DNA polymerase 0.5μL, 25mmol / L MgCl 2 2 μL, 2 μL of 10 mmol / L dNTP, 5 μL of 5×PCR amplification buffer, 2 μL of DNA template, and 10.5 μL of sterile deionized water. Multiplex PCR amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 57°C for 50 s, extension at 72°C for 60 s, and 35 cycles of amplification; finally, extension...
Embodiment 3
[0041] The detection of embodiment 3 multiplex PCR system in the artificial pollution sample
[0042] The 10-fold dilution method was used to carry out 10-fold serial dilution of the overnight-cultured Listeria monocytogenes ST121 strain, and sequentially obtained 2.5×10 8 cfu / mL, 2.5×10 7 cfu / mL, 2.5×10 6 cfu / mL, 2.5×10 5 cfu / mL, 2.5×10 4 cfu / mL, 2.5×10 3 cfu / mL, 2.5×10 2 cfu / mL, 2.5×10 1 cfu / mL, 2.5×100cfu / mL, take 1mL concentration as 2.5×10 4 cfu / mL, 2.5×10 3 cfu / mL, 2.5×10 2 cfu / mL, 2.5×10 1 Cfu / mL, 2.5×100cfu / mL Listeria monocytogenes ST121 strain was added to 9mL of milk, then added to 90mL of BHI medium and cultured for 4h, 6h, 8h, 10h and 12h, and then extracted by bacterial genome Kit for genomic DNA extraction. Take 2 μL of genomic DNA as a template for multiplex PCR reactions. Multiplex PCR uses 25 μL reaction system, including: 0.5 μL each of multiplex PCR primers 0609-F, 0609-R, 0171-F, 0171-R, prfA-F and prfA-R at a concentration of 10 μmol / L, 2.5 U / ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More