A test kit for human staphylococcus detection

A staphylococcus and kit technology, applied in the direction of microorganism-based methods, microorganism measurement/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 Effects of low and high specificity

Inactive Publication Date: 2020-08-28
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 human staphylococci from other staphylococci due to the lack of specific primers, so joint generation sequencing is required to Identification, high cost, takes several days to get results
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 test kit for human staphylococcus detection
  • A test kit for human staphylococcus detection
  • A test kit for human staphylococcus detection

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 human Staphylococcus rpoB gene sequence, 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 Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus wahlschii, Staphylococcus capitis, 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 epidermidis, Staphylococcus wattleri, Staphylococcus capitum, and Staphylococcus hemolyticus. Therefore, Cas12a protein cannot target To Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus wahlschii, Staphylococcus capitus, Staphylococcus hemolyticus, therefore, only human staphylococcus can show positive reaction during detection, and other stap...

Embodiment 2

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

[0019] (1) Prepare human Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus worbachii, Staphylococcus capitis, Staphylococcus haemolyticus 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 TwistAmpbasic kit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R ( Sequence is SEQ No.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 (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 human Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus worbachii, Staphylococcus capitis, Staphylococcus haemolyticus 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 TwistAmpbasic kit reaction drying ball (TwistDx company), 0.9 μl 10mM RPA-F (sequence is SEQ No.2) and RPA-R ( Sequence is SEQ No.3), 0.375 μl Ribonuclease Inhibitor (Takara Company), 3.5 μl buffer2.1 (NEB Company), 1000nM guide RNA (sequence is SEQ No...

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Abstract

The invention provides a kit for detecting staphylococcus hominis. The kit comprises a guide RNA specifically targeting a staphylococcus hominis rpoB gene, amplification primer pairs, hydrated TwistAmp basic kit reaction drying balls, an LbCas12a protein, a single-stranded DNA probe (ssDNA), a Ribonuclease Inhibitor and a buffer solution. The kit is used for detecting the staphylococcus hominis, the detection specificity is high, and the staphylococcus hominis can be distinguished from other staphylococcocci including staphylococcus aureus, staphylococcus epidermidis, staphylococcus warneri, staphylococcus capitis and staphylococcus haemolyticus. The RNA sequence is used for detection, the consumed time is about 1 hour, no PCR instrument is needed, the requirements for equipment are low and significantly lower than those of a traditional bacterial culture method or PCR sequencing method, and the clinical practical 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 human staphylococcus, comprising a guide RNA specifically targeting the rpoB gene of human staphylococcus, a pair of RPA amplification primers, and a hydrated TwistAmp basic kit reaction Drying balls, LbCas12a protein, single-stranded DNA probe (ssDNA), Ribonuclease Inhibitor, and buffer. This kit can be used for rapid detection of human Staphylococcus. Background technique [0002] Staphylococcus hominis is a common bacterial group among Gram-positive 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 human staphylococci from other staphylococci due to the lack of specific primers, so joint generation sequencing is required to Identification is expensive ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12R1/44
CPCC12Q1/6844C12Q1/689C12Q2522/101C12Q2563/107C12Q2531/119
Inventor 孙泽玮陈文静郑良荣
Owner ZHEJIANG UNIV
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