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Quantitative detection method and quantitative detection kit for anti-radiation acinetobacter and application of quantitative detection kit

A quantitative detection method and quantitative detection technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the time-consuming and labor-intensive detection cost of Acinetobacter radioresistant, which cannot meet the requirements of fast and accurate , Low-cost detection of radiation-resistant Acinetobacter and other issues, to achieve good microbial species specificity, fast detection speed, and strong specificity

Pending Publication Date: 2021-06-22
SHANGHAI INST OF TECH
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is: the traditional method of physiological and biochemical detection of Acinetobacter radiation-resistant is time-consuming and labor-intensive and the detection cost is high, and cannot meet the needs of fast, accurate and low-cost detection of Acinetobacter radiation-resistant.

Method used

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  • Quantitative detection method and quantitative detection kit for anti-radiation acinetobacter and application of quantitative detection kit
  • Quantitative detection method and quantitative detection kit for anti-radiation acinetobacter and application of quantitative detection kit

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Embodiment 1

[0031] The present embodiment provides the method for making the Acinetobacter radioresistens ATCC 43998 RT-PCR standard curve, which specifically includes the following steps:

[0032] (1) Extract the total RNA of Acinetobacter radiation-resistant ATCC 43998, measure the absorbance value (260nm) by UV spectrophotometer, measure the total RNA concentration to be 920ng / μL, and calculate the colony according to the genome size of Acinetobacter radiation-resistant ATCC 43998 The number is 2.26×10 8 CFU / mL;

[0033] (2) Reverse transcribe the above-mentioned RNA to obtain cDNA, do 10-fold gradient dilution, and perform fluorescence quantitative PCR amplification reaction with the diluted cDNA template; take the logarithm of the positive template of cDNA with different copy numbers as the abscissa, Taking the initial cycle number (Ct value) reaching the fluorescence threshold in the fluorescence quantitative PCR reaction process as the ordinate to obtain the RT-PCR standard curve ...

Embodiment 2

[0037] A quantitative detection method for Acinetobacter radioresistens, comprising the following steps:

[0038] (1) Prepare 5 strains to be tested and sterilized water negative control substance for testing. The 5 strains to be tested are 1# Lactococcus lactis IL1403, 2# Acinetobacter radioresistant a, 3# Lactobacillus plantarum B21, 4 #Acinetobacter baumannii, and 5#Radioresistant Acinetobacter b;

[0039] (2) Extract the total RNA in the above-mentioned strains to be tested respectively, and then reverse-transcribe it into cDNA, and use the respective cDNA as a template to perform fluorescent quantitative PCR amplification using a specific primer pair for specifically amplifying Acinetobacter radioresistant;

[0040] Wherein the primer pair of specific amplification radiation-resistant Acinetobacter is: upstream primer, its sequence is shown in SEQ ID No: 1; Downstream primer, its sequence is shown in SEQ ID No: 2; Above-mentioned fluorescent quantitative PCR amplification...

Embodiment 3

[0045] A radiation-resistant Acinetobacter quantitative detection kit, including RNA extraction reagents, RNA reverse transcription reagents, wherein the RNA extraction reagents use Trizol kits, namely Plus RNA Purification System, U.S. Invitrogen Company, article number 12183-555; wherein said reverse transcription reagent adopts reverse transcription kit, i.e. M-MLV RTasecDNA Synthesis Kit, Dalian Bao Biological Engineering Co., Ltd., article number D6130; also includes PCR amplification Amplification system, described PCR amplification system adopts quantitative PCR kit, namely PrimeScript TM RT-PCR Kit, Dalian Bao Biological Engineering Co., Ltd., article number DRR063A; also includes upstream detection primers and downstream detection primers, wherein the sequence of the upstream detection primers is shown in SEQ ID No: 1, and the sequence of the downstream detection primers is as shown in Shown in SEQ ID No: 2; also includes a quantitative external standard, the quant...

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Abstract

The invention discloses a quantitative detection method and a quantitative detection kit for anti-radiation acinetobacter and application of the quantitative detection method and the quantitative detection kit. The method comprises the following steps: extracting total RNA in a sample to be detected; carrying out reverse transcription on the total RNA to obtain cDNA; carrying out fluorescent quantitative PCR amplification on the cDNA and then detecting a Ct value; and substituting the Ct value into a pre-fitted standard curvilinear equation so as to calculate the number of the acinetobacter radians in the sample. The kit comprises an RNA extraction reagent, an RNA reverse transcription reagent, a PCR amplification reaction reagent, a specific primer pair for amplifying anti-radiation acinetobacter and a PCR quantitative external standard substance. The detection method has the characteristics of rapidness, strong specificity, high sensitivity, low cost and the like.

Description

technical field [0001] The invention relates to a quantitative detection method, quantitative detection kit and application of radiation-resistant Acinetobacter, belonging to the technical field of biological detection. Background technique [0002] As the interface with the external environment, the skin is an important barrier against the invasion of pathogens. Microorganisms of all kinds gather on the surface of the skin: bacteria, fungi and viruses. Under the influence of the external environment that the epidermis contacts, skin microorganisms form their unique and complex flora structure. From the perspective of biodiversity, microorganisms such as Propionibacterium acnes, Staphylococcus, Corynebacterium, and Malassezia together constitute the diversity of skin microorganisms. Most of these microorganisms are symbionts that compete with invading pathogenic microorganisms. In some cases, excessive reproduction or excessive deficiency of a certain type of microbial fl...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/06C12N15/11
CPCC12Q1/6851C12Q2600/166C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 王彩霞王玥马来记施雁勤周亮李学娇
Owner SHANGHAI INST OF TECH
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