Reagent kit for detecting enterobacter aerogenes

A technology of Enterobacter aerogenes and kits, which is applied in the field of kits for the detection of Enterobacter aerogenes, can solve the problems of high cost, delay in disease treatment, lack of specific primers, etc., and achieve the effect of low equipment requirements and high specificity

Inactive Publication Date: 2019-09-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection method is bacterial culture or combined PCR sequencing, but bacterial culture takes 5-7 days, and PCR often cannot distinguish Enterobacter aerogenes from other Enterobacter bacteria due to the lack of specific primers, so combined Only gen

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  • Reagent kit for detecting enterobacter aerogenes
  • Reagent kit for detecting enterobacter aerogenes
  • Reagent kit for detecting enterobacter aerogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Sequence Alignment of the rpoB Gene of Enterobacter Bacteria

[0015] Such as figure 1 As shown, in the rpoB gene sequence of Enterobacter aerogenes, the 5' end of the guide RNA sequence (selected part) of the present invention is the PAM structure of TTTN. The guide RNA sequence does not have a completely consistent sequence in Enterobacter cloacae, Enterobacter Jagovii, and Enterobacter sakazakii, and there are several base differences. Moreover, there is no PAM structure of TTTN at the 5' end of the homologous sequences in Enterobacter cloacae, Enterobacter jaegavii, and Enterobacter sakazakii. Therefore, using this guide RNA, the Cas12a protein cannot be targeted to Enterobacter cloacae, Enterobacter cloacae, and Enterobacter sakazakii. Enterobacter sakazakii and Enterobacter sakazakii, therefore, only Enterobacter aerogenes can show a positive reaction during the test, and the rest of the Enterobacter genus bacteria can show a negative reaction, which c...

Embodiment 2

[0016] Embodiment 2: The guide RNA specificity determination of the present invention, the steps are as follows:

[0017] (1) Prepare standard strains of Enterobacter aerogenes, Enterobacter cloacae, Enterobacter Jagovius, and Enterobacter sakazakii;

[0018] (2) Take 1ml of the sample to be tested, heat it at 98 degrees Celsius for 5min, and draw 1μl as the test sample;

[0019] (3) Prepare the reaction system, the reaction system is 25 μl, including 1 μl detection sample, 14.75 μl hydrated TwistAmp basickit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R ( Sequence is SEQNo.3), 0.375 μl Ribonuclease Inhibitor (Takara company), 3.5 μl buffer2.1 (NEB company), 1000nM guide RNA (sequence is SEQ No.1), 250nM Cas12a (NEB company), 200nM single-stranded DNA probe Needle (Shanghai Sangong), add water to make up to 25μl;

[0020] (4) React at a constant temperature of 37 degrees Celsius for 30 minutes;

[0021] (5) After the reaction, pl...

Embodiment 3

[0023] Embodiment 3: the detection method based on guide RNA described in the present invention and traditional PCR method specificity and experimental time-consuming comparison

[0024] 1. based on the detection method of guide RNA described in the present invention, the steps are as follows:

[0025] (1) Prepare standard strains of Enterobacter aerogenes, Enterobacter cloacae, Enterobacter Jagovius, and Enterobacter sakazakii;

[0026] (2) Take 1ml of the sample to be tested, heat it at 98 degrees Celsius for 5min, and draw 1μl as the test sample;

[0027] (3) Prepare the reaction system, the reaction system is 25 μl, including 1 μl detection sample, 14.75 μl hydrated TwistAmp basickit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R ( Sequence is SEQNo.3), 0.375 μl Ribonuclease Inhibitor (Takara company), 3.5 μl buffer2.1 (NEB company), 1000nM guide RNA (sequence is SEQ No.1), 250nM Cas12a (NEB company), 200nM single-stranded DNA p...

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Abstract

The invention provides a reagent kit for detecting enterobacter aerogenes. The reagent kit contains a guide RNA specifically targeted to an enterobacter aerogenes rpoB gene, an amplification primer pair, a hydration TwistAmp basic kit reaction drying ball, LbCas12a protein, a single-stranded DNA probe (ssDNA), a Ribonuclease inhibitor and a buffer solution. When the reagent kit is used for detecting the enterobacter aerogenes, the detection specificity is high, and the enterobacter aerogenes can be distinguished from other enterobacter bacteria including enterobacter cloacae, enterobacter gergoviae and enterobacter sakazakii. Besides, when the RNA sequence is used for detection, time consumption is about 1 hour, a PCR instrument is not needed, requirements for equipment are low and are notably lower than those by a traditional bacteria culture method or a PCR sequencing method, and the clinical use value is high.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic bacteria, and relates to a kit for detection of Enterobacter aerogenes. This kit can be used for rapid detection of Enterobacter aerogenes. Background technique [0002] Enterobacter aerogenes is a common bacterial group among Gram-negative bacteria that infects humans, and it brings a huge burden to patients and society. The traditional detection method is bacterial culture or combined PCR sequencing, but bacterial culture takes 5-7 days, and PCR often cannot distinguish Enterobacter aerogenes from other Enterobacter bacteria due to the lack of specific primers, so combined It can only be identified by first-generation sequencing, which is costly and takes several days to produce results. During this period, clinicians can only use medicines based on experience because they do not obtain test results, which often leads to delays in disease treatment. Therefore, how to quickly detect Ente...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2527/127C12Q2563/107Y02A50/30
Inventor 孙泽玮陈文静郑良荣
Owner ZHEJIANG UNIV
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