A kit for detecting Enterobacter cloacae

A technology of Enterobacter cloacae and a kit, which is applied in the field of kits for the detection of Enterobacter cloacae, can solve the problems of high cost, delay in disease treatment, and inability to distinguish Enterobacter cloacae from other Enterobacter genus bacteria, and achieve low equipment requirements, highly specific effect

Inactive Publication Date: 2020-04-10
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 cloacae from other Enterobacter bacteria due to the lack of specific primers, so combined generation is required Sequencing can only be identified, the cost is high, and it takes several days to get the result
During this period, clinicians can only use medicines based on experience because they have not obtained test results, which often leads to delays in disease treatment

Method used

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  • A kit for detecting Enterobacter cloacae
  • A kit for detecting Enterobacter cloacae
  • A kit for detecting Enterobacter cloacae

Examples

Experimental program
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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 cloacae, 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 among Enterobacter aerogenes, Enterobacter Jagovii, and Enterobacter sakazakii, but there are several base differences. Moreover, there is no PAM structure of TTTN at the 5' end of the homologous sequences in Enterobacter aerogenes, Enterobacter Jagovii, and Enterobacter sakazakii. Therefore, using this guide RNA, the Cas12a protein cannot be targeted to Enterobacter aerogenes, Enterobacter aerogenes, and Enterobacter sakazakii. Enterobacter jagovius and Enterobacter sakazakii, therefore, only Enterobacter cloacae can show a positive reaction during the test, and the rest of the Enterobacter genus bacteria can show a negative reaction, wh...

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 cloacae, Enterobacter aerogenes, 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 TwistAmpbasickit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R (sequence (SEQ No.3), 0.375 μl Ribonuclease Inhibitor (Takara Company), 3.5 μl buffer2.1 (NEB Company), 1000 nM guide RNA (sequence is SEQ No.1), 250 nM Cas12a (NEB Company), 200 nM 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 cloacae, Enterobacter aerogenes, 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 TwistAmpbasickit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R (sequence (SEQ No.3), 0.375 μl Ribonuclease Inhibitor (Takara Company), 3.5 μl buffer2.1 (NEB Company), 1000 nM guide RNA (sequence is SEQ No.1), 250 nM Cas12a (NEB Company), 200 nM single-stranded DNA p...

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Abstract

The invention provides a kit for the detection of Enterobacter cloacae, including guide RNAs specifically targeting the rpoB gene of the Enterobacter cloacae, a primer pair for amplification, hydrated TwistAmp basic kit reaction drying balls, LbCas12a proteins, a single-stranded DNA probe (ssDNA), Ribonuclease Inhibitor and buffer. The kit can be used to detect the Enterobacter cloacae with high specificity, and can distinguish the Enterobacter cloacae from other Enterobacter bacteria, including Enterobacter aerogenes, Enterobacter gergoviae and Enterobacter sakazakii. At the same time, the method using the RNA sequence described in the invention for detection takes about 1 hour, does not need any PCR instruments, and has low requirements for equipment; and time consumption of the method is significantly lower than that of the conventional bacterial culture method or the PCR sequencing method, with great clinical practical value.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic bacteria, and relates to a kit for detection of Enterobacter cloacae. This kit can be used for rapid detection of Enterobacter cloacae. Background technique [0002] Enterobacter cloacae is a common bacterial group among Gram-negative bacteria that infects human beings, which 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 cloacae from other Enterobacter bacteria due to the lack of specific primers, so combined generation is required Sequencing can only be identified, the cost is high, and it takes several days to get the 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 En...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107Y02A50/30
Inventor 孙泽玮陈文静郑良荣
Owner ZHEJIANG UNIV
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