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A kind of RT-PCR kit for detecting Brucella amikacin drug resistance and its application method

A technology of RT-PCR and amikacin, applied in the biological field, can solve problems affecting the treatment of patients with brucellosis, and achieve the effect of promoting diagnosis

Active Publication Date: 2022-06-21
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Amikacin is used as a drug for the treatment of brucellosis in some areas. If the strain of Brucella infected by the patient is resistant to amikacin, it will seriously affect the treatment of brucellosis.

Method used

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  • A kind of RT-PCR kit for detecting Brucella amikacin drug resistance and its application method
  • A kind of RT-PCR kit for detecting Brucella amikacin drug resistance and its application method
  • A kind of RT-PCR kit for detecting Brucella amikacin drug resistance and its application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Assembly of RT-PCR Detection Kit

[0045] (1) Prepare RT-PCR reaction solution

[0046] a. Design specific amplification primers and probes and send them to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis. The specific primer sequences are:

[0047] seq ID No.1: 5'-AATGCGATCAAGTCGGGCG-3'

[0048] seq ID No.2: 5'-TGCCATCATAAAGGCCGGTG-3'

[0049] seq ID No.3: 5'FAM-TGCCATCATAAAGGCCGGTG-3'BHQ-1

[0050] seq ID No.4: 5'-AACTGTTCGCCAGGCTCAAG-3'

[0051] seq ID No.5: 5'-AAGCGGCCATTTTCCACCAT-3'

[0052] seq ID No.6: 5'VIC-TCGTGACCCATGGCGATGCCTGCTT-3'BHQ-2

[0053] b. Preparation of RT-PCR solution:

[0054] Described A liquid includes RT-PCR buffer, dNTP liquid, Mg 2+ Solution RT-DNA polymerase and sterilized double-distilled water, the B solution includes specific primer pairs seq ID No. 1 and seq ID No. 2 and probe seq ID No. 3 for detecting Brucella, and Specific primer pairs seq ID No.4 and seq ID No.5 and probe seq ID No.6 for detection of amikacin resist...

Embodiment 2

[0067] Embodiment 2 Brucella amikacin drug resistance RT-PCR kit detects minimum bacterial content

[0068] (1) After streaking the amikacin-resistant Brucella strain for 48 hours, adjust the turbidity of the strain to 0.5McF (about 1×10 8 CFU / mL). Take 1 μL of bacterial solution for 10-fold serial dilution, and the final low bacterial content is 10 5 -10 1 CFU / μL.

[0069] (2) Use a genome extraction kit to extract the genomic DNA of the sample to be tested, and the nucleic acid concentration detector detects that the concentration is higher than 1 ng / μL.

[0070] (3) Using the extracted DNA of the sample to be tested as a template, the template, solution A and solution B are mixed in proportion, and at the same time, a negative quality control standard is compared, and RT-PCR amplification is performed.

[0071] The fluorescence quantitative amplification program was: 95°C for 30s, 95°C for 5s, 60°C for 30s, 40 cycles.

[0072] The PCR amplification system is 25 μL / samp...

Embodiment 3

[0075] Embodiment 3 Test kit detects the unknown Brucella strain of amikacin

[0076] The RT-PCR kit of the present invention is used to detect the unknown Brucella strain of amikacin, and a negative control is set.

[0077] For the feasibility results of the kit to detect amikacin unknown Brucella, see figure 2 . The fluorescence amplification curve shows that there is no amplification curve for the negative quality control standard, and there are amplification curves for the FAM and VIC signals of the positive quality control standard, and the CT value is 34.16 (consistent with the CT value between 32-35), indicating that This test comparison was established. The test results show that sample 1 is an amikacin-resistant Brucella strain, and its FAM and VIC signals both have amplification curves; sample 2 is an amikacin-resistant dilution sample, which is a 10-fold dilution of sample 1 Sample (manual dilution operation); Sample 3 is an amikacin-sensitive Brucella strain, t...

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Abstract

An RT-PCR kit for detecting Brucella amikacin drug resistance and a method for using the same belong to the field of biotechnology. The kit includes liquid A (RT-PCR buffer, RT-DNA polymerase and sterilized double distilled water), liquid B (primer pair, probe), negative quality control standard and positive quality control standard. Including specific primer pair seq ID No.1 and seq ID No.2 and probe seq ID No.3 for detecting Brucella, and specific primers for detecting amikacin resistance of Brucella For seq ID No.4 and seq ID No.5 and probe seq ID No.6. The RT-PCR kit is practical, time-consuming, and reproducible. After RT-PCR amplification, it can quickly, effectively and intuitively identify whether clinical samples contain amikacin-resistant brucea. bacteria.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a RT-PCR kit for detecting Brucella amikacin drug resistance and a method for using the same Background technique [0002] Brucella is a facultative intracellular parasite that belongs to the "B class bioterrorism agent", which causes persistent infection of the host by parasitizing the immune system of the host, and can cause global zoonotic diseases— Brucellosis (hereinafter referred to as brucellosis). Brucellosis affects public health safety, mainly manifested as fever, sweating, fatigue, joint pain, etc. In severe cases, patients lose their ability to work and ultimately affect economic development [1]. The "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" classifies brucellosis, SARS, anthrax, AIDS, and rabies as Class B infectious diseases. The common Brucella species that cause human brucellosis infection are sheep (B.m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2600/106C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 杨晓雯姜海赵鸿雁朴东日田国忠范玉
Owner ICDC CHINA CDC
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