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Kit for rapidly detecting acinetobacter baumannii and using method

A technology of Acinetobacter baumannii and a kit, which is applied in the field of kits for rapid detection of Acinetobacter baumannii, can solve the problems of high price, long time-consuming isolation and identification of Acinetobacter baumannii, and achieve easy operation and good promotion Application prospect, obvious effect of technical effect

Pending Publication Date: 2020-11-03
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a kind of test kit and using method for rapidly detecting Acinetobacter baumannii, and the described test kit and using method for rapidly detecting Acinetobacter baumannii need to solve The technical problems of time-consuming and expensive methods for isolating and identifying Acinetobacter baumannii in the prior art

Method used

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  • Kit for rapidly detecting acinetobacter baumannii and using method
  • Kit for rapidly detecting acinetobacter baumannii and using method
  • Kit for rapidly detecting acinetobacter baumannii and using method

Examples

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Effect test

Embodiment 1

[0035] Example 1 Primer Design

[0036] The basic principles of primer design in the LAMP reaction system include that the Tm value of the outer primer set should be 55-63°C, and the base length should be 15-25; the Tm value of about 20 bases at the 5' end of the inner primer set should be higher than other primer sets. The Tm value of the primer set is controlled at 60-68°C, and the Tm value of about 20 bases at the 3' end of the internal primer set is at 55-63°C; the GC content of all primers is controlled at 30%-65%, and it is necessary to ensure that the 5 ' and 3' end stability is required to avoid dimer formation between the primers themselves and between the primers. There are strict requirements on the position of the primers anchored on the target sequence. The base length between the external primer sets is controlled within 160-220, and the base between the corresponding internal primers and external primers is controlled within 20. The base length between groups i...

Embodiment 2

[0039] Example 2 Sensitivity analysis of loop-mediated isothermal amplification detection kit for Acinetobacter baumannii

[0040] (1) Extraction and detection of Acinetobacter baumannii genomic DNA

[0041]Genomic DNA of Acinetobacter baumannii (standard strain ATCC 19606, derived from American Type Culture Collection ATCC) was extracted using a bacterial genomic DNA extraction kit (purchased from Beijing Suo Laibao Technology Co., Ltd.). And use the ScanDrop100 nucleic acid analyzer to detect the concentration of Acinetobacter baumannii genomic DNA, calculate the copy number of the DNA template by the molecular weight, then dilute the DNA template to 10 with double distilled water 1 ~10 10 Copy number / 2 μL. , and respectively take 2 μL as a template for LAMP amplification.

[0042] (2) prepare 25 μ L system in PCR tube, this system comprises 8U / μ L Bst polymerase, LAMP buffer solution, primer set (each primer concentration is as described in claim 3), template DNA 2 μ L ...

Embodiment 3

[0045] Example 3 Specific detection of loop-mediated isothermal amplification detection kit for Acinetobacter baumannii

[0046] Acinetobacter baumannii (Aba), Haemophilus influenzae (Hin), Klebsiella pneumoniae (Kpn), Pseudomonas aeruginosa (Pae), Staphylococcus aureus (Sau), Escherichia coli (Eco) , Streptococcus pneumoniae (Spn), Moraxella catarrhalis (Mac) are common respiratory tract infection bacteria. Clinically, rapid differential diagnosis of the pathogenic bacteria of bacterial pneumonia is of great significance. The standard bacterial strains mentioned above were all obtained from the American Type Culture Collection ATCC. The above-mentioned standard strains were enriched according to routine and then used for later use.

[0047] (1) For the extraction of sample DNA, DNA was extracted separately using the bacterial genome DNA extraction kit (purchased from Beijing Suo Lai Bao Technology Co., Ltd.) extraction kit instructions.

[0048] (2) Establish a LAMP reacti...

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Abstract

The invention relates to a kit for rapidly detecting acinetobacter baumannii. The kit comprises an upstream external primer f3 of which the sequence is shown as SEQ ID NO: 1, a downstream external primer b3 of which the sequence is shown as SEQ ID NO: 2, an upstream internal primer fip of which the sequence is shown as SEQ ID NO: 3, a downstream internal primer bip of which the sequence is shown as SEQ ID NO: 4, and an upstream cyclization primer lf of which the sequence is shown as SEQ ID NO: 5. The primer group in the kit disclosed by the invention is designed according to specific genes 16SrRNA and 23S rRNA of acinetobacter baumannii and a spacer sequence (GenBank ID: KY659326.1) of the specific genes 16S rRNA and 23S rRNA, and is proved to have good specificity by verification. The kit disclosed by the invention is simple and convenient to operate, high in reaction speed, high in sensitivity (400 copies per milliliter) and controllable in condition and is suitable for popularization in medical institutions at all levels.

Description

technical field [0001] The invention belongs to the field of biological detection and relates to a kit, in particular to a kit for rapid detection of Acinetobacter baumannii and a use method thereof. Background technique [0002] Acinetobacter baumannii (Acinetobacter baumannii) is the most common Gram-negative bacillus in the genus Acinetobacter, which can infect many parts including respiratory system, urinary system, wound, abdominal cavity and nervous system. It has strong acquired drug resistance, which brings difficulties to the application of broad-spectrum antibiotics, and is also one of the main causes of nosocomial infections in all countries in the past three decades. Acquired pneumonia is the main infection caused by this pathogen, while infections involving the central nervous system, skin, and soft tissues have also emerged, posing a serious threat to medical institutions. [0003] At present, the identification methods of Acinetobacter baumannii include isola...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 范列英司玉莹李根叶扬琴张雯雁陆柳
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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