A rapid detection method for bacteria in complex samples
A detection method and complex sample technology, applied in the field of molecular biology, can solve the problems of limited scalability, high price, difficult one-time use, etc., and achieve the effect of simple experimental steps, flexible and simple operation, and low cost
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Embodiment 1
[0043] Example 1 Analysis of Pathogenic Escherichia coli O157:H7 (CICC 10907) in Human Whole Blood Samples Using a Nanoporous Hydrogel-Based Loop-Mediated Isothermal Amplification Method
[0044] 1. The primer sequence is as follows:
[0045] F3: 5'-GCCATCTCCTGATGACGC-3';
[0046] B3: 5'-ATTTACCGCAGCCAGACG-3';
[0047] LF: 5'-CTTTGTAACAACCTGTCATCGACA-3';
[0048] LB: 5'-ATCAATCTCGATATCCATGAAGGTG-3';
[0049] FIP: 5'-CATTTTGCAGCTGTACGCTCGCAGCCCATCATGAATGTTGCT-3';
[0050] BIP: 5'-CTGGGGCGAGGTCGTGGTATTCCGACAAACACCACGAATT-3';
[0051] 2. The actual original DNA concentrations of Escherichia coli in human whole blood samples a to e are:
[0052] Human whole blood sample a: The original concentration of E. coli DNA is 1 CFU / μL;
[0053] Human whole blood sample b: the original concentration of E. coli DNA is 10 CFU / μL
[0054] Human whole blood sample c: the original concentration of Escherichia coli DNA is 20 CFU / μL;
[0055] Human whole blood sample d: the original concen...
Embodiment 2
[0062] Example 2 Analysis of Listeria monocytogenes (CICC 21633) in milk tea samples by a ring-mediated isothermal amplification method based on nanoporous hydrogel
[0063] 1. The primer sequence is as follows:
[0064] F3: 5'-TTGCGCAACAAACTGAAGC-3';
[0065] B3: 5'-GCTTTTACGAGAGCACCTGG-3';
[0066] LF: 5'-TAGGACTTGCAGGCGGAGATG-3';
[0067] LB: 5'-GCCAAGAAAAGGTTACAAAGATGG-3';
[0068] FIP: 5'-CGTGTTTCTTTTCGATTGGCGTCTTTTTTTCATCCATGGCACCACC-3';
[0069] BIP: 5'-CCACGGAGATGCAGTGACAAATGTTTTGGATTTCTTCTTTTTCTCCACAAC-3';
[0070] 2. Add 1×LAMP buffer, 6mM MgSO to 0.83μL, 1.67μL and 2.50μL milk tea samples respectively 4 , 1.4mM dNTP, 640U / mL Bst 2.0 DNA polymerase, 1.6μM FIP and BIP, 0.2μM F3 and B3, 0.8μM LF and LB, 1mg / mL BSA, 0.1mg / mL lysozyme, 1X EvaGreen, 2.0μL bacteria Liquid or DNA solution, 1.6mg Four-arm PEGAcrylate and 1.1mg SH-PEG-SH were used to prepare the LAMP reaction hydrogel system mixture (25μL) with 3 different milk tea sample concentrations.
[0071] 3. Th...
Embodiment 3
[0073] Example 3 Analysis of Salmonella typhi (CICC 10871) in bovine whole blood samples by a loop-mediated isothermal amplification method based on nanoporous hydrogel
[0074] 1. The primer sequence is as follows:
[0075] F3: 5'-GGCGATATTGGTGTTTATGGGG-3';
[0076] B3: 5'-AACGATAAACTGGACCACGG-3';
[0077] LF: 5'-GACGAAAGAGCGTGGTAATTAAC-3';
[0078] LB: 5'-GGGCAATTCGTTATTGGCGATAG-3';
[0079] FIP: 5'-GACGACTGGTACTGATCGATAGTTTTTCAACGTTTCCTGCGG-3';
[0080] BIP: 5'-CCGGTGAAATTATCGCCACACAAAACCCACCGCCAGG-3'.
[0081] 2. Add 1×LAMP buffer, 6mM MgSO to 0.5μL, 1.0μL and 2.0μL bovine whole blood samples respectively 4 , 1.4mM dNTP, 640U / mL Bst 2.0 DNA polymerase, 1.6μM FIP and BIP, 0.2μM F3 and B3, 0.8μM LF and LB, 1mg / mL BSA, 0.1mg / mL lysozyme, 1X EvaGreen, 2.0μL bacteria Liquid or DNA solution, 1.6mg Four-arm PEGAcrylate and 1.1mg SH-PEG-SH prepared three LAMP-reactive hydrogel system mixtures (25μL) with different bovine whole blood sample concentrations.
[0082] 3. The sa...
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