Kit for detecting staphylococcus capitis

A staphylococcus and kit technology, applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as high cost, delay in disease treatment, lack of specific primers, etc., and meet equipment requirements Low and high specificity effects

Inactive Publication Date: 2019-08-16
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 Staphylococcus capitis from other staphylococci due to the lack of specific primers, so joint generation sequencin

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  • Kit for detecting staphylococcus capitis
  • Kit for detecting staphylococcus capitis
  • Kit for detecting staphylococcus capitis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Staphylococcus rpoB gene sequence alignment

[0017] like figure 1 As shown, in the rpoB gene sequence of Staphylococcus capitis, 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 Staphylococcus aureus, Staphylococcus human, Staphylococcus wahlschii, Staphylococcus epidermidis, and Staphylococcus hemolyticus, and all have several base differences. In addition, there is no PAM structure of TTTN at the 5' end of the homologous sequence in Staphylococcus aureus, Staphylococcus human, Staphylococcus watterii, Staphylococcus epidermidis, and Staphylococcus hemolyticus. Therefore, the Cas12a protein cannot be targeted by using this guide RNA. To Staphylococcus aureus, Staphylococcus hominis, Staphylococcus wahlschii, Staphylococcus epidermidis, and Staphylococcus hemolyticus, therefore, only Staphylococcus capitis can show a posi...

Embodiment 2

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

[0019] (1) Prepare Staphylococcus capitis, Staphylococcus aureus, Staphylococcus hominis, Staphylococcus worbachii, Staphylococcus epidermidis, Staphylococcus hemolyticus standard strains;

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

[0021] (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;

[0022] (4) React at a constant temperature of 37 degrees Celsius...

Embodiment 3

[0025] Embodiment 3: the detection method based on guide RNA described in the present invention and traditional PCR method specificity and experimental time-consuming comparison

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

[0027] (1) Prepare Staphylococcus capitis, Staphylococcus aureus, Staphylococcus hominis, Staphylococcus worbachii, Staphylococcus epidermidis, Staphylococcus hemolyticus standard strains;

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

[0029] (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), 25...

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Abstract

The invention provides a kit for detecting staphylococcus capitis. The kit comprises guide RNA specifically targeting staphylococcus capitis rpoB gene, an amplification primer pair, hydration TwistAmpbasic kit reaction drying balls, LbCas12a protein, a single-chain DNA probe (ssDNA), a Ribonuclease Inhibitor and a buffer solution. When the kit is used for detecting the staphylococcus capitis, high detection specificity is achieved, and the staphylococcus capitis can be distinguished from other staphylococcus such as staphylococcus aureus, staphylococcus hominis, staphylococcus warneri, staphylococcus epidermidis and staphylococcus haemolyticus. Meanwhile, when the RNA sequence is used to perform detection, detection time is about 1 hour, a PCR instrument is not needed, equipment requirements are evidently lower than those of a traditional bacterial culture method or a PCR sequencing method, and a high clinical practical value is achieved.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic bacteria, and relates to a kit for the detection of Staphylococcus capitus, comprising a guide RNA specifically targeting the rpoB gene of Staphylococcus capitis, a pair of RPA amplification primers, and a hydrated TwistAmp basickit Reaction dry balls, LbCas12a protein, single-stranded DNA probe (ssDNA), Ribonuclease Inhibitor, and buffer. This kit can be used for rapid detection of Staphylococcus capitis. Background technique [0002] Staphylococcus capitis is a common bacterial group among Gram-positive bacteria that infects humans, 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 Staphylococcus capitis from other staphylococci due to the lack of specific primers, so joint generation sequencing is required It can only be identified, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12R1/44
CPCC12Q1/6844C12Q1/689C12Q2521/301C12Q2527/127C12Q2563/107
Inventor 孙泽玮陈文静郑良荣
Owner ZHEJIANG UNIV
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