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Super bacteria steno fluorescent quantitative PCR detection kit

A detection kit, super bacteria technology, applied in the direction of determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., to achieve the effect of good linear relationship, good specificity and low false positives

Active Publication Date: 2019-01-15
ANHUI DAJIAN MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since the superbug Steno appeared not long ago, and the sequencing of the superbug Steno genome has just been completed, there is no report on the detection of the superbug Steno by real-time fluorescent quantitative PCR technology. Therefore, a fluorescent quantitative PCR for the detection of the superbug Steno is designed. The detection kit is of great significance for the early detection and clinical treatment of superbugs

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  • Super bacteria steno fluorescent quantitative PCR detection kit
  • Super bacteria steno fluorescent quantitative PCR detection kit
  • Super bacteria steno fluorescent quantitative PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: be used for the design and the screening of the primer and the probe that are detected to superbug Steno fluorescence quantitative PCR

[0037] According to the genomic DNA sequence of the superbug Steno recorded in GenBank (GenBank: CP011305.1), the conserved sequence of Steno 23s rRNA was selected, and TaqMan probes and primers were designed and synthesized. Avoid regions with high homology with other pathogens, manually adjust parameters, design multiple sets of alternative primers and probes, select FAM (5′ end) as the fluorescent reporter group for the fluorescent label of the probe, TAMRA (3′ end) ) as a fluorescence quencher. Screen alternative primers and probes. The screening principle of primers / probes is: select primers and probes with higher PCR amplification efficiency and probe binding efficiency in the target conserved region as candidate primers and probes.

[0038] In this embodiment, 20 groups of forward primers, reverse primers and flu...

Embodiment 2

[0043] Embodiment 2: be used for the specificity detection of the primer and the probe that the superbug Steno fluorescent quantitative PCR detects

[0044] The present embodiment adopts superbug Steno, Staphylococcus epidermidis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Candida tropicalis, Pseudomonas aeruginosa, Klebsiella pneumoniae bacteria, Candida albicans, Candida glabrata, Acinetobacter baumannii, Acinetobacter calcoacetate, Acinetobacter jonesi, Enterococcus faecium, Enterococcus faecalis, Stenotrophomonas maltophilia The primers and probes screened in Example 1 were tested for specificity.

[0045] Genomic DNA of the above-mentioned bacteria was extracted respectively, and the primers and probes screened in Example 1 were used as templates to perform real-time fluorescence quantitative PCR.

[0046] The fluorescence quantitative reaction was carried out on ABI 7900 detector, the reaction system was 25 μL, forward a...

Embodiment 3

[0061] Embodiment 3: Optimization of the reaction system and reaction conditions of superbug Steno fluorescent quantitative PCR detection

[0062] 1. Optimization of the reaction system

[0063] 1.1 Optimization of primer and probe concentration

[0064] The concentration of primers is a key factor affecting the PCR reaction. If the concentration is too low, the reaction will be incomplete; if there are too many primers, the possibility of mismatching and non-specific products will be greatly increased. Non-specific products and primer dimers also compete with the target sequence for DNA polymerase and dNTP substrates, thereby reducing the amplification of the target sequence.

[0065] The concentration of the probe mainly determines the level of background fluorescence, and the level of background fluorescence is related to the sensitivity of the reaction system.

[0066] Mix forward primer (20 μM) and reverse primer (20 μM) at a volume ratio of 1:1, add 0.2 μL, 0.3 μL, 0.4...

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Abstract

The invention discloses primers and a probe for superbacteria Steno fluorescence quantitative PCR (polymerase chain reaction) detection. The sequence of the forward primer is disclosed as SEQ ID NO.1, the sequence of the reverse primer is disclosed as SEQ ID NO.2, and the sequence of the probe is disclosed as SEQ ID NO.3; and the 5' end of the probe is marked with a fluorescence report group, and the 3' end is marked with a fluorescent quenching group. The invention also discloses a superbacteria Steno detection kit and detection method. The specific primers and fluorescent probe are designed for the superbacteria Steno, thereby enhancing the detection sensitivity and specificity and having low false positive rate. The detection kit disclosed by the invention has the advantages of high detection sensitivity and favorable specificity.

Description

technical field [0001] The invention relates to a super bacteria Steno fluorescent quantitative PCR detection kit, which belongs to the technical field of bacterial molecular biology detection. Background technique [0002] "Super bacteria" (super bacteria) refers to some drug-resistant bacteria that most effective antibiotics cannot defeat. When the human body is infected by this "super bacteria", medical treatment is often difficult, and it is difficult to choose effective antibiotics. "Super bacteria" is easy to spread in medical and health institutions, and its consequences often lead to the death of patients, thus causing panic and anxiety about "super bacteria". In recent years, with the widespread use of antibacterial drugs, anti-tumor chemotherapy drugs and immunosuppressants, the situation of bacterial drug resistance in the world has become increasingly severe. At present, the "super drug-resistant bacteria" considered by infectious disease experts mainly include:...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2561/101
Inventor 钟明李香梅
Owner ANHUI DAJIAN MEDICAL TECH CO LTD