Kit for detecting staphylococcus warneri

The technology of a Staphylococcus wornerii and a kit is applied in the field of kits for the detection of Staphylococcus wornerii, which can solve the problems of high cost, delay in disease treatment, lack of specific primers, etc., and achieves high specificity and low equipment requirements. Effect

Inactive Publication Date: 2019-08-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection method is to carry out bacterial culture or combined PCR sequencing, but bacterial culture takes 5-7 days, and PCR often cannot distinguish Staphylococcus wausii from other staphylococci due to the lack of specific primers, so joint generation sequencing is required 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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  • Kit for detecting staphylococcus warneri
  • Kit for detecting staphylococcus warneri
  • Kit for detecting staphylococcus warneri

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Sequence alignment of Staphylococcus rpoB gene

[0017] Such as figure 1 As shown, in the rpoB gene sequence of Staphylococcus voornii, 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 identical sequence among Staphylococcus aureus, Staphylococcus human, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus haemolyticus, and all have several base differences. And the homologous sequences in Staphylococcus aureus, Staphylococcus human, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus haemolyticus have no PAM structure of TTTN at the 5'end. Therefore, using this guide RNA, Cas12a protein cannot be targeted To Staphylococcus aureus, Staphylococcus human, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hemolyticus, therefore, only Staphylococcus wokerii can show a positive reaction during the test, and ...

Embodiment 2

[0018] Example 2: The specific determination of the guide RNA of the present invention, the steps are as follows:

[0019] (1) Prepare standard strains of Staphylococcus wokerii, Staphylococcus aureus, Staphylococcus human, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hemolyticus;

[0020] (2) Take 1ml of the sample to be tested, heat it at 98°C for 5min, and draw 1μl as the test sample;

[0021] (3) Prepare the reaction system, the reaction system is 25μl, including 1μl test sample, 14.75μl hydration TwistAmp basickit reaction dry ball (TwistDx company), 0.9μl 10mM RPA-F (sequence SEQ No. 2) and RPA-R ( The sequence is SEQ No. 3), 0.375μl Ribonuclease Inhibitor (Takara), 3.5μl buffer2.1 (NEB), 1000nM guide RNA (SEQ No.1), 250nM Cas12a (NEB), 200nM single-stranded DNA probe Needle (Shanghai Shenggong), add water to make up to 25μl;

[0022] (4) Constant temperature reaction at 37 degrees Celsius for 30 minutes;

[0023] (5) After the reaction is over, place the P...

Embodiment 3

[0025] Example 3: Comparison of specificity and experimental time-consuming of the detection method based on the guide RNA of the present invention and the traditional PCR method

[0026] 1. Based on the guide RNA detection method of the present invention, the steps are as follows:

[0027] (1) Prepare standard strains of Staphylococcus wokerii, Staphylococcus aureus, Staphylococcus human, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hemolyticus;

[0028] (2) Take 1ml of the sample to be tested, heat it at 98°C for 5min, and draw 1μl as the test sample;

[0029] (3) Prepare the reaction system, the reaction system is 25μl, including 1μl test sample, 14.75μl hydration TwistAmp basickit reaction dry ball (TwistDx company), 0.9μl 10mM RPA-F (sequence SEQ No. 2) and RPA-R ( The sequence is SEQ No. 3), 0.375μl Ribonuclease Inhibitor (Takara), 3.5μl buffer2.1 (NEB), 1000nM guide RNA (SEQ No.1), 250nM Cas12a (NEB), 200nM single-stranded DNA probe Needle (Shanghai Sheng...

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Abstract

The present invention provides a kit for detecting staphylococcus warneri. The kit comprises a guide RNA specifically targeting a staphylococcus warneri rpoB gene, an amplification primer pair, hydration TwistAmp basic kit reaction drying spheres, LbCas12a protein, a single-stranded DNA probe (ssDNA), an ribonuclease inhibitor and a buffer solution. The kit is used for detecting the staphylococcuswarneri and high in detection specificity, and can separate the staphylococcus warneri from other staphylococci, including staphylococcus aureus, staphylococcus hominis, staphylococcus epidermidis, staphylococcus capitis and staphylococcus haemolyticus. At the same time, the use of the RNA sequence for the detection takes about 1 hour, does not require a PCR instrument, has low requirements on equipment, and is significantly lower than a conventional bacterial culture method or a PCR sequencing method and large in clinical practical value.

Description

Technical field [0001] The invention belongs to the field of pathogen detection, and relates to a kit for the detection of Staphylococcus woernerii, comprising a guide RNA that specifically targets the rpoB gene of Staphylococcus woerneri, RPA amplification primer pair, and hydration TwistAmp basickit Reaction dry ball, LbCas12a protein, single-stranded DNA probe (ssDNA), Ribonuclease Inhibitor and buffer. This kit can be used for rapid detection of Staphylococcus wokerii. Background technique [0002] Staphylococcus wokerii is a common group of gram-positive bacteria in human infections, which brings a huge burden to patients and society. The traditional detection method is bacterial culture or PCR combined sequencing, but bacterial culture requires 5-7 days, and PCR often lacks specific primers and cannot distinguish Staphylococcus wocheni from other Staphylococci, so combined first-generation sequencing is required It can be identified, the cost is high, and it takes several...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/44
CPCC12Q1/689
Inventor 孙泽玮陈文静郑良荣
Owner ZHEJIANG UNIV
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