CN116144660B - MRSA bacterial detection based on novel CRISPR target - Google Patents

MRSA bacterial detection based on novel CRISPR target Download PDF

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CN116144660B
CN116144660B CN202310047377.1A CN202310047377A CN116144660B CN 116144660 B CN116144660 B CN 116144660B CN 202310047377 A CN202310047377 A CN 202310047377A CN 116144660 B CN116144660 B CN 116144660B
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CN116144660A (en
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杨海燕
王瑛
杜悦
段广才
陈帅印
晋乐飞
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Zhengzhou University
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Abstract

The invention provides crRNA1 which can recognize a target point shown as SEQ ID NO.1 of clfA, and crRNA2 which can recognize a target point shown as SEQ ID NO.2 of mecA. The invention also provides a composition composed of crRNA1 and crRNA2, a CRISPR-based kit and further detection application thereof. The invention obtains the identification targets of clfA and mecA genes through repeated screening, thereby successfully realizing sensitive and specific detection of MRSA.

Description

MRSA bacterial detection based on novel CRISPR target
Technical Field
The invention relates to a detection reagent and a method based on a new target for detecting MRSA.
Background
Staphylococcus aureus (golden wine bacteria) is an important human opportunistic pathogen that can cause a variety of diseases such as: endocarditis, pneumonia, toxic shock syndrome, etc. Penicillin findings have led to effective treatment of patients infected with staphylococcus aureus, but Methicillin Resistant Staphylococcus Aureus (MRSA) soon emerged with large area use of antibiotics. It is therefore highly necessary to construct an easy to handle rapid screening test for MRSA. The CRISPR-Cas system provides a revolutionary tool for genome editing, and Cas proteins with alternative activities become a powerful tool for detecting nucleic acid sensitivity. Recent studies have shown the potential to utilize these new CRISPR-Cas technologies to provide low cost and practical diagnostic tools for pathogen and disease detection.
Disclosure of Invention
In one aspect, the invention provides crRNA 1 which can recognize a target point shown as SEQ ID NO.1 of a staphylococcus aureus specific recognition gene clfA or has a sequence shown as SEQ ID NO. 3.
In one aspect, the invention provides crRNA 2 which can recognize a target point shown as SEQ ID NO.2 of a methicillin-resistant gene mecA or has a sequence shown as SEQ ID NO. 4.
Correspondingly, the invention provides a crRNA composition which is a combination of crRNA 1 and crRNA 2 and can be used for detecting MRSA staphylococcus aureus.
In a further aspect, the invention provides a kit comprising the crRNA composition described above, which may contain Cas12a protein, which may be referred to as CRISPR-Cas12a based kit. The kit can contain a single-stranded DNA fluorescent probe, and the structure can be 5'6-FAM-TTTTTTTTTTTT-3' BHQ1.
Correspondingly, the invention provides a method for detecting MRSA by using the gene or the combined application form thereof, the method can be used for non-diagnosis purpose, whether a sample contains methicillin resistant genes or drug-resistant staphylococcus aureus or not can be judged, and the sample can be a clinically isolated staphylococcus aureus specimen. Detection may include pre-subjecting the sample to clfA, mecA gene amplification, which may be isothermal amplification, such as RAA recombinase-mediated isothermal nucleic acid amplification, primer a for amplifying the staphylococcus aureus clfA gene, which may comprise an upstream primer ATGAATATGAAGAAAAAAGAAAAACACGCAATTC and a downstream primer ACGCTACTTGAATCATTACTTTTGCTTTCGTTAC; primer B for amplifying the methicillin-resistant gene mecA may comprise an upstream primer CCCAATTTTGATCCATTTGTTGTTTGATATAGTCTTCAGA and a downstream primer GAATGCAGAAAGACCAAAGCATACATATTGAAAA.
The beneficial effects of the invention are as follows: the identification targets of clfA and mecA genes are obtained through repeated screening, and further sensitive and specific detection of MRSA is successfully realized.
Drawings
FIG. 1 shows the results of clfA gene-specific assay
FIG. 2 shows the results of mecA gene-specific assays
FIG. 3 shows the sample identification results of example 2.
Detailed Description
The following examples are directed to the following genes:
1. The staphylococcus aureus specific recognition gene clfA recognizes a target SEQ ID NO.1: TTTTGGACTACTCAGCAGTAAAGACRRNA SEQ ID NO.3: UAAUUUCUACUAAGUGUAGAUGGACUACUCAGCAGUAAAGARAA isothermal amplification primer:
An upstream primer: ATGAATATGAAGAAAAAAGAAAAACACGCAATTC A
A downstream primer: ACGCTACTTGAATCATTACTTTTGCTTTCGTTAC A
2. Methicillin-resistant gene mecA, recognition target SEQ ID NO.2: TTTCGGTCTAAAATTTTACCACGTCRRNA SEQ ID NO.4: UAAUUUCUACUAAGUGUAGAUGGUCUAAAAUUUUACCACGURAA isothermal amplification primer:
an upstream primer: CCCAATTTTGATCCATTTGTTGTTTGATATAGTCTTCAGA A
A downstream primer: GAATGCAGAAAGACCAAAGCATACATATTGAAAA A
3. Fluorescent probe: 5'6-FAM-TTTTTTTTTTTT-3' BHQ1
Example 1:
The clfA gene and mecA gene in MRSA were detected using CRISPR-Cas12 a-bound RAA isothermal amplification technique, and in order to facilitate calculation of gene copy numbers, sensitivity detection was performed using synthetic plasmids containing clfA gene and mecA gene, respectively, and further specificity detection was performed using laboratory-preserved bacteria. The method specifically comprises the following steps:
(1) Nucleic acid amplification was performed on the clfA gene and mecA gene using the RAA nucleic acid amplification reagent using the sample genomic DNA (DNA of synthetic plasmid for sensitivity detection and DNA of bacteria stored in the laboratory for bacterial experiments) as a template. The reaction system comprises 50ul of components: 41.5ul A Buffer,2.5ul B Buffer,2ul sample DNA,2ul upstream primer, 2ul downstream primer.
(Primer sequence: clfA gene upstream primer SEQ ID NO.5: ATGAATATGAAGAAAAAAGAAAAACACGCAATTC, downstream primer SEQ ID NO.6: ACGCTACTTGAATCATTACTTTTGCTTTCGTTAC;
The primer upstream of mecA gene SEQ ID NO.7: CCCAATTTTGATCCATTTGTTGTTTGATATAGTCTTCAGA, downstream primer SEQ ID NO.8: GAATGCAGAAAGACCAAAGCATACATATTGAAAA)
The specific operation steps are as follows:
a. 41.5ul of A Buffer, 2.0ul of upstream primer (10 uM) and 2.0ul of downstream primer (10 uM) were added to a test cell tube containing the dry powder enzyme preparation.
B. 2.0ul of sample DNA (plasmid DNA or bacterial DNA) was added to the test cell tube.
C. Then 2.5ul of B Buffer is added into the detection unit tube, and the mixture is uniformly mixed and centrifuged at low speed for 10 seconds.
D. the detection unit tube is placed in a constant temperature incubator or a constant temperature water bath kettle at 37 ℃ for incubation for 30 minutes, and then amplification products are obtained.
E. After the reaction, taking 5-10ul of reaction system for electrophoresis detection, using phenol: chloroform: isoamyl alcohol (25:24:1) is used for extracting and purifying the reaction solution in a volume ratio of 1:1, the reaction solution is centrifuged at 12000rpm/min for 3-5 minutes, and the supernatant is taken for spot detection. Sequencing the purified product, wherein the sequencing result is as follows:
The clfA amplification sequence was:
CGTGTAAATCGATTGGCGTGGCTTCAGTGCTTGTAGGTACGTTAATCGGTTTTGGACTACTCAGCAGTAAAGAAGCAGATGCAAGTGAAAATAGTGTTACGCAATCTGATAGCGCAAGTAACGAAAGCAAAAGTAATGATTCAAGTAGCGTAATA
The mecA amplification sequence is:
TCATACTTAGTTCTTTAGCGATTGCTTTATAATCTTTTTTAGATACATTCTTTGGAACGATGCCTATCTCATATGCTGTTCCTGTATTGGCCAATTCCACATTGTTTCGGTCTAAAATTTTACCACGTTCTGATTTTAAATTTTCAATATGTATGCTTTGGTCTTTCTGCATTCA
BLAST comparison of the sequenced Gene sequence with the Gene sequence on the Gene bank shows that: clfA is 100% identical to Staphylococcus aureus strain pt217 chromosomeme, complex genome; mecA is 100% identical to Staphylococcus aureus strain pt217 chromosomeme, complex genome.
(2) The clfA gene and mecA gene were detected separately. The detection system comprises 50ul of: 5ul (the amplification product of the previous step) of amplified product, 5ul of Cas12a (1 pM) protein, 5ul of buffer, 5ul of single-stranded DNA fluorescent probe (10 uM), 2.5ul of crRNA (the crRNA) and 27.5ul of enzyme-free water. And placing the detection system in a 96-well culture dish, placing the culture dish in an enzyme-labeled instrument, detecting the fluorescence intensity with excitation light 494nm and emission light 520nm, detecting once every 2min, and continuously detecting at 37 ℃ for 60min to obtain a continuous fluorescence value report.
( CrRNA sequence: clfA: UAAUUUCUACUAAGUGUAGAUGGACUACUCAGCAGUAAAGA, mecA: UAAUUUCUACUAAGUGUAGAUGGUCUAAAAUUUUACCACGU A )
Detection result:
(1) Sensitivity of
In evaluating the sensitivity of the method, synthetic plasmids were selected for experiments to facilitate calculation of gene copy numbers. The synthesized plasmids are diluted according to the concentration gradient, the concentration is 10 -5,10-4,10-3,10-2,10-1,10-0 copies in sequence, and the amplification detection is carried out in sequence according to the method, and the result is as follows: the method can detect 10 -3 copies of the gene to be detected; only when the corresponding crRNA, cas12a protein, fluorescent probe and positive gene are added simultaneously, the fluorescence value can be detected.
2) Specificity of the sample
We performed simultaneous detection with laboratory-preserved Staphylococcus epidermidis as in FIG. 1, sample 2 was laboratory-preserved Staphylococcus aureus, clearly distinguished from Staphylococcus epidermidis. As shown in fig. 2, sample 1, sample 2 and sample 3 are methicillin-resistant staphylococcus aureus preserved in a laboratory, sample 4 and sample 5 are methicillin-sensitive staphylococcus aureus, and the two are obviously distinguished.
Example 2:
1. Sample collection
The bacterial sample used in the experiment was a clinically isolated staphylococcus aureus.
2. Experimental method
(1) Preparation of DNA templates
Whole genome DNA was extracted from bacterial samples using a DNA kit. Taking out the bacterial sample freezing tube from the refrigerator at-80 ℃, re-heating the tube in the refrigerator at 4 ℃ for 5 hours, quickly dipping the bacterial liquid in an ultra-clean bench by adopting a sterile inoculating loop, inoculating the tube on a Columbia blood agar culture plate by a sectional streaking method, incubating the tube for 20 hours at 37 ℃ in a constant temperature box, picking up single bacterial colony on the blood plate, inoculating the bacterial colony in brain heart immersion liquid culture medium, and carrying out shaking culture at 37 ℃ for 16 hours. 2ml of bacterial liquid is taken and operated according to the instruction.
(2) RAA isothermal nucleic acid amplification
Nucleic acid amplification was performed on the clfA gene and mecA gene using RAA nucleic acid amplification reagents, respectively. The reaction system comprises 50ul of components: 41.5ul A Buffer,2.5ul B Buffer,2ul sample DNA,2ul upstream primer, 2ul downstream primer.
A. 41.5ul of A Buffer, 2.0ul of upstream primer (10 uM) and 2.0ul of downstream primer (10 uM) were added to a test cell tube containing the dry powder enzyme preparation.
B. 2.0ul of sample DNA was added to the test cell tube.
C. Then 2.5ul of B Buffer is added into the detection unit tube, and the mixture is uniformly mixed and centrifuged at low speed for 10 seconds.
D. the detection unit tube is placed in a constant temperature incubator or a constant temperature water bath kettle at 37 ℃ for incubation for 30 minutes, and then amplification products are obtained.
(3) CRISPR-Cas12a detection
Detection of clfA gene and mecA gene, respectively:
the detection system comprises 50ul of: 5ul of amplified product, 5ul of Cas12a (1 pM) protein, 5ul of buffer, 5ul of single-stranded DNA fluorescent probe (10 uM), 2.5ul of crRNA, and 27.5ul of enzyme-free water. And placing the detection system in a 96-well culture dish, placing the culture dish in an enzyme-labeled instrument, detecting the fluorescence intensity with excitation light 494nm and emission light 520nm, detecting once every 2min, and continuously detecting at 37 ℃ for 60min to obtain a continuous fluorescence value report.
3. Detection result
As shown in FIG. 3, the clfA gene and mecA gene were detected positively at the same time, and it was determined that methicillin-resistant Staphylococcus aureus was detected.

Claims (9)

  1. CrRNA 1 can recognize a target point shown as SEQ ID NO.1 of a MRSA staphylococcus aureus specific recognition gene clfA, and the sequence of the crRNA 1 is shown as SEQ ID NO. 3.
  2. CrRNA 2 can identify a target point shown as SEQ ID NO.2 of a methicillin-resistant gene mecA, and the sequence of the crRNA 2 is shown as SEQ ID NO. 4.
  3. 3. A kit for CRISPR-based detection of MRSA staphylococcus aureus comprising the crRNA 1 of claim 1, the crRNA 2 of claim 2, cas12a protein, and a single-stranded DNA fluorescent probe.
  4. 4. The kit of claim 3, wherein the kit comprises a single-stranded DNA fluorescent probe having the structure 5'6-FAM-TTTTTTTTTTTT-3' bhq1.
  5. 5. The kit of claim 3, further comprising reagents required for amplification of the clfA or mecA genes.
  6. 6. The kit of claim 5, wherein the amplification is isothermal amplification.
  7. 7. The kit of claim 6, wherein the isothermal amplification is RAA recombinase-mediated isothermal nucleic acid amplification.
  8. 8. The kit according to any one of claims 3 to 7, wherein the kit further comprises amplification primers A and/or B, the sequences of the upstream and downstream primers of the amplification primer A are shown in SEQ ID NO.5-6, and the sequences of the upstream and downstream primers of the amplification primer B are shown in SEQ ID NO. 7-8.
  9. 9. A method for detecting MRSA staphylococcus aureus for non-diagnostic purposes, comprising detecting MRSA bacteria using the kit of any one of claims 3-8.
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CN110684823A (en) * 2019-10-23 2020-01-14 海南大学 Test strip-based microorganism rapid diagnosis technology for Cas12a enzyme
CN111378722A (en) * 2019-11-04 2020-07-07 江苏大学 Specific nucleic acid fragment nano-fluorescence trace rapid detection method based on CRISPR-Cas12g
CN111676272A (en) * 2020-07-03 2020-09-18 上海交通大学 Cas12 a/crRNA-based food-borne pathogenic bacteria detection method

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