Primer and/or probe composition for detecting cocci causing bloodstream infections and application of primer and/or probe composition
A composition and probe technology, applied in the field of molecular biology, can solve the problems of long cycle, increased cost of bacterial resistance treatment, and difficulty for clinicians to choose a treatment plan.
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Embodiment 1
[0112] This example provides the composition of exemplary kits.
[0113] Specifically, an exemplary kit includes reagent bottles or tubes respectively containing the following components, and a packaging box for collectively packaging these reagent bottles or tubes.
[0114] 1) Nucleic acid extraction solution: composed of 2% (M / V) Chelex-100, 1% (V / V) Tris-HCl, 1M, pH9.0;
[0115] 2) PCR reaction enzyme system: 5U / μL Taq DNA polymerase and 2U / μL uracil-N-glycosylase (UNG enzyme) mixed at a volume ratio of 3:1;
[0116] 3) Internal standard: a plasmid containing the internal standard int gene fragment;
[0117] 4) Negative control substance: double distilled water purified by Millipore water purifier;
[0118] 5) Positive controls: T vector plasmid carrying the target gene for Streptococcus pneumoniae (lytA), T vector plasmid carrying the target gene for Enterococcus faecalis (Ef0027), T vector plasmid carrying the target gene for Enterococcus faecium (ddl), carrying T vect...
Embodiment 2
[0131] This example provides a method for establishing an assay using the exemplary kit in Example 1.
[0132] Specifically, the method includes the following steps:
[0133] 1) Preparation of reference substances: Take 50 μL each of the positive control substance and negative control substance, place them in 1.5mL (or 0.5mL) centrifuge tubes (shake and mix for 10 seconds after the frozen reagent has melted), add 50 μL of nucleic acid extraction solution to fully Mix well, incubate at 98°C for 10 min, then centrifuge at 12,000 rpm for 5 min, take 2 μL of supernatant for PCR reaction.
[0134] 2) Preparation of the reaction system mixture: 16 μL of each primer-probe mixture was mixed with 0.2 μL of the PCR reaction enzyme system, oscillated for several seconds, and centrifuged at 3000 rpm for several seconds.
[0135] 3) PCR amplification: Add 2 μL each of the treated supernatants of the negative control substance and the positive control substance into the PCR tubes of the re...
Embodiment 3
[0141] This example provides specificity experiments performed on the kits and methods of the present application.
[0142] 1) Experimental materials
[0143] The experimental materials used in the following experiments and their sources are listed below:
[0144]
[0145]
[0146] 2) Experimental method
[0147] Using the experimental method established in Example 2, the above-mentioned 12 kinds of pathogenic bacteria were detected respectively, so as to analyze and evaluate the specificity of the kit.
[0148] 3) Experimental results
[0149] Utilize the kit in the embodiment 1 to detect 12 kinds of common pathogenic bacteria (diphtheria, Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, Proteus, Proteus mirabilis, Enterobacter cloacae, Streptococcus sanguis, albicans, Klebsiella oxytoca, Burkholderia cepacia, Acinetobacter baumannii), the results were all negative (see Table 1).
[0150] Table 1 The specificity verification results of the kit of th...
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