CN110129463B - Kit for detecting staphylococcus capitis - Google Patents
Kit for detecting staphylococcus capitis Download PDFInfo
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- CN110129463B CN110129463B CN201910370161.2A CN201910370161A CN110129463B CN 110129463 B CN110129463 B CN 110129463B CN 201910370161 A CN201910370161 A CN 201910370161A CN 110129463 B CN110129463 B CN 110129463B
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention provides a kit for detecting staphylococcus capitis, which comprises guide RNA of a specific targeted staphylococcus capitis rpoB gene, an amplification primer pair, a hydrated twist amp basic kit reaction dry sphere, LbCas12a protein, a single-stranded DNA probe (ssDNA), a Ribonuclose Inhibitor and buffer solution. The kit is used for detecting the staphylococcus caput, has high detection specificity, and can distinguish the staphylococcus caput from other staphylococcus including staphylococcus aureus, human staphylococcus, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus haemolyticus. Meanwhile, the RNA sequence of the invention is used for detection, the time consumption is about 1 hour, a PCR instrument is not needed, the requirement on equipment is low, the method is obviously lower than that of a traditional bacterial culture method or a PCR sequencing method, and the clinical practical value is high.
Description
Technical Field
The invention belongs to the field of pathogenic bacteria detection, and relates to a kit for detecting staphylococcus capitis, which comprises guide RNA of a specific targeted staphylococcus capitis rpoB gene, an RPA amplification primer pair, a hydrated twist Amp basic reaction dry sphere, LbCas12a protein, a single-stranded DNA probe (ssDNA), a Ribonucleae Inhibitor and buffer solution. The kit can be used for quickly detecting the staphylococcus capitis.
Background
Staphylococcus capitis is a common flora in gram-positive bacteria infected by human bodies, and brings huge burden to patients and society. The traditional detection method is to carry out bacterial culture or PCR combined sequencing, but the bacterial culture needs 5-7 days, and the PCR often cannot distinguish staphylococcus capitis from other staphylococcus due to lack of specific primers, so that the identification can be carried out only by combined first-generation sequencing, the cost is high, and the result can be obtained only in several days. During this period, the clinician can only administer the drug empirically because no test results are obtained, often resulting in delayed treatment of the disease. Therefore, how to rapidly detect the staphylococcus capitis is very important. Recently, Zhang Feng et al found that the LbCas12a protein can be used for detecting pathogenic bacteria, and the detection can be completed in about 1 hour, so that the time and the labor are saved, and the sensitivity is high. The specific principle is that the system comprises LbCas12a protein, pathogenic bacteria DNA, guide RNA and single-stranded DNA (ssDNA) with a fluorescent group and a quenching group at two ends respectively. Wherein, the DNA sequence of the pathogenic bacteria needs to contain a PAM structure of TTTN, and the sequence needs to have specificity, namely to be distinguished from other pathogenic bacteria. The guide RNA sequence is a sequence behind TTTN in the specific target sequence of the pathogenic bacteria, and the length of the guide RNA sequence is 20-24 basic groups. Under the guidance of guide RNA, LbCas12a protein can be combined to a pathogen-specific DNA sequence, and then LbCas12a protein can show Dnase activity, so that single-stranded DNA with a fluorescent group and a quenching group at two ends is cut, the fluorescent group and the quenching group are separated, fluorescence is generated, and the existence of a pathogen is indicated.
Disclosure of Invention
The invention aims to provide a kit for detecting staphylococcus capitis, which comprises guide RNA specifically targeting staphylococcus capitis rpoB gene, an RPA amplification primer pair, a hydrated twist amp basic kit reaction dry ball, LbCas12a protein, a single-stranded DNA probe (ssDNA), a Ribonucleae Inhibitor and buffer solution. The guide RNA of the specific targeting staphylococcus capilalis rpoB gene has a nucleotide sequence shown in SEQ No.1, can specifically target staphylococcus capilalis but cannot target staphylococcus aureus, human staphylococcus, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus haemolyticus, and can be used for detecting staphylococcus capilalis. The sequence of the RPA amplification primer pair RPA-F and RPA-R is shown in SEQ No.2 and SEQ No. 3. The sequence of the single-stranded DNA probe is shown in SEQ No. 6.
The invention also aims to provide a method for rapidly detecting staphylococcus capitis based on the kit, which is realized by the following steps:
the method comprises the following specific steps: 1ml of the sample was heated at 95-98 ℃ for 5min, 1. mu.l of the sample was used as a test sample, and 14.75. mu.l of hydrated twist Amp basic kit reaction dry spheres (twist Dx Co.), 0.9. mu.l of 10mM RPA-F (SEQ ID NO: 2) and RPA-R (SEQ ID NO: 3), 0.375. mu.l of Ribonuclase Inhibitor (Takara Co.), 3.5. mu.l of buffer2.1(NEB Co.), 1000nM guide RNA (SEQ ID NO: 1), 250nM Cas12a (NEB Co.), 200nM single-stranded DNA probe (Shanghai Biolabs, SEQ ID NO: 6) were added with water to make up to 25. mu.l. Reacting at 37 ℃ for 25-45min, and detecting staphylococcus capitis by directly observing the fluorescence change of a detection system under a fluorescence microscope; the yellow-green fluorescence indicates that the staphylococcus capitis is polluted, and the non-luminescence indicates that the staphylococcus capitis is not polluted. The method can distinguish Staphylococcus capitis from other staphylococci, including Staphylococcus aureus, Staphylococcus hominis, Staphylococcus wovensis, Staphylococcus epidermidis, and Staphylococcus haemolyticus.
Wherein:
the single-stranded DNA probe ssDNA sequence (SEQ No.6) is: 5 '6-FAM-TTATT-3' BHQ1, synthesized by Shanghai.
The detection kit for detecting staphylococcus capitis infection has the advantages that:
(1) compared with the traditional bacterial culture or PCR combined one-generation sequencing method, the traditional method needs several days, and the method can complete detection in 1 hour. Meanwhile, the method adopts the RPA method for amplification, the reaction can be completed only by a constant-temperature water bath kettle, a PCR instrument is not needed, and the requirement on equipment is low.
(2) When the detection kit is used for detection, only the staphylococcus capitis is in a positive reaction, and other staphylococcus, including common staphylococcus aureus, human staphylococcus, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus haemolyticus, are in negative reactions. Therefore, the detection kit has high specificity.
Drawings
FIG. 1 is a comparison of rpoB gene sequences of Staphylococcus capitis, Staphylococcus aureus, Staphylococcus hominis, Staphylococcus wovensis, Staphylococcus epidermidis and Staphylococcus haemolyticus.
FIG. 2 shows the result of fluorescence microscopy, wherein the staphylococcus strains, such as staphylococcus capitis, staphylococcus aureus, staphylococcus hominis, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus haemolyticus, are added from tube Nos. 1-6. Wherein, tube No.1 has yellow green fluorescence and shows positive reaction. No. 2-6 tubes have no yellow green fluorescence and show negative reaction.
FIG. 3 shows that different species of staphylococci were amplified by PCR method to obtain bands with similar sizes, and head-shaped staphylococci could not be distinguished.
FIG. 4 is a graph showing the comparison of the time consumption of the experiment using the detection method based on the guide RNA of the present invention and the conventional PCR sequencing method in combination.
Detailed Description
The invention is further described below with reference to the figures and examples.
Example 1: alignment of staphylococcal rpoB Gene sequences
As shown in FIG. 1, in the sequence of the staphylococcus capitis rpoB gene, 5' end of the guide RNA sequence (selected part) of the invention is PAM structure of TTTN. The guide RNA sequence has no completely consistent sequence in staphylococcus aureus, staphylococcus hominis, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus haemolyticus, and has a difference of a plurality of bases. And 5' ends of homologous sequences in staphylococcus aureus, staphylococcus hominis, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus haemolyticus are all free of a PAM structure of TTTN, so that by using the guide RNA, the Cas12a protein cannot target the staphylococcus aureus, staphylococcus hominis, staphylococcus epidermidis and staphylococcus haemolyticus, and only the staphylococcus capitis can show positive reaction while the rest of the staphylococcus is negative reaction during detection. As shown in FIG. 1, the 5' end of the sequence (in the box-selected part of the figure) is a PAM sequence of TTTN structure on the rpoB gene of Staphylococcus capitis, and the sequence is not found in Staphylococcus aureus, Staphylococcus hominis, Staphylococcus wovensis, Staphylococcus epidermidis, and Staphylococcus haemolyticus, which are commonly found in Staphylococcus. In addition, in other staphylococci, the 5' end of the homologous sequence of the sequence does not contain a PAM structure of TTTN, so that the sequence can specifically target staphylococcus capitis, but cannot target staphylococcus aureus, staphylococcus hominis, staphylococcus wowensis, staphylococcus epidermidis and staphylococcus haemolyticus, and can be used for specific detection of staphylococcus capitis.
Example 2: the guide RNA specificity determination method comprises the following steps:
(1) preparing standard strains of staphylococcus capitis, staphylococcus aureus, human staphylococcus, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus haemolyticus;
(2) taking 1ml of a sample to be detected, heating at 98 ℃ for 5min, and sucking 1 mu l of the sample to be detected as a detection sample;
(3) preparing a reaction system, the reaction system being 25. mu.l comprising 1. mu.l of a test sample, 14.75. mu.l of hydrated twist Amppasic kit reaction dry spheres (twist Dx Co.), 0.9. mu.l of 10mM RPA-F (SEQ No.2) and RPA-R (SEQ No.3), 0.375. mu.l of Ribonucleae Inhibitor (Takara Co.), 3.5. mu.l of buffer2.1(NEB Co.), 1000nM guide RNA (SEQ No.1), 250nM Cas12a (NEB Co.), 200nM single-stranded DNA probe (Shanghai Producer), and adding water to 25. mu.l;
(4) reacting at 37 ℃ for 30 min;
(5) after the reaction is finished, the PCR tube is placed under a fluorescence microscope for direct observation, the existence of staphylococcus capitis is proved by fluorescence, and the absence of fluorescence indicates the pollution of staphylococcus capitis.
As shown in FIG. 2, the result of the detection was positive in the case where the sample 1 was a Staphylococcus capitis and fluorescence was observed. The rest samples are non-cephalic staphylococci, have no fluorescence and show negative reaction.
Example 3 detection of guide RNA based on the invention compared with the conventional PCR method in specificity and time consuming experiments
1. The method for detecting the guide RNA based on the invention comprises the following steps:
(1) preparing standard strains of staphylococcus capitis, staphylococcus aureus, human staphylococcus, staphylococcus wovensis, staphylococcus epidermidis and staphylococcus haemolyticus;
(2) taking 1ml of a sample to be detected, heating at 98 ℃ for 5min, and sucking 1 mu l of the sample to be detected as a detection sample;
(3) preparing a reaction system, the reaction system being 25. mu.l comprising 1. mu.l of a test sample, 14.75. mu.l of hydrated twist Amppasic kit reaction dry spheres (twist Dx Co.), 0.9. mu.l of 10mM RPA-F (SEQ No.2) and RPA-R (SEQ No.3), 0.375. mu.l of Ribonucleae Inhibitor (Takara Co.), 3.5. mu.l of buffer2.1(NEB Co.), 1000nM guide RNA (SEQ No.1), 250nM Cas12a (NEB Co.), 200nM single-stranded DNA probe (Shanghai Producer), and adding water to 25. mu.l;
(4) reacting at 37 ℃ for 30 min;
(5) after the reaction is finished, the PCR tube is placed under a fluorescence microscope for direct observation, the existence of staphylococcus capitis is proved by fluorescence, and the pollution of staphylococcus capitis is shown by the absence of fluorescence;
(6) as shown in FIG. 2, the result of the detection was positive in the case where the sample 1 was a Staphylococcus capitis and fluorescence was observed. The rest samples are non-cephalic staphylococci, have no fluorescence and show negative reaction.
2. The traditional PCR method comprises the following experimental steps:
(1) designing a primer, wherein the sequence of the PCR primer is 20 bases initiated at the 5' end of the RPA primer, so that the obtained PCR product has the same sequence with the RPA product, and the sequence of a primer pair is shown in SEQ No.4 and SEQ No. 5;
(2) taking 1ml of a sample to be detected, heating at 98 ℃ for 5min, and sucking 1 mu l of the sample to be detected as a detection sample;
(3) preparing a reaction system, wherein the reaction system comprises 12.5 mul of DNA polymerase premix (Shanghai works), 1 mul of forward primer, 1 mul of reverse primer, 1 mul of sample to be detected and 9.5 mul of water;
(4) carrying out PCR reaction;
(5) performing agarose gel electrophoresis;
(6) as shown in FIG. 3, 188bp fragments were amplified from all samples, and it was impossible to distinguish Staphylococcus capitis from other Staphylococcus species.
As mentioned above, the PCR method cannot distinguish the species of staphylococci, so sequencing comparison is required, the experiment cost is high, the time is long, and the disease condition may be delayed.
As shown in FIG. 4, the detection method using the detection kit of the present invention has high specificity and takes a short time of only about 1 hour, while the PCR method is used, since the sequences of different species of staphylococci are highly homologous, the difference in a sequence is often only a few bases, the designed primers can be amplified by all strains, the obtained products are close in size and cannot be distinguished by agarose gel, and thus the sequencing is required, and the experiment takes about 2 days. The method of the invention takes significantly less time than the PCR group.
Sequence listing
<110> Zhejiang university
<120> kit for detecting staphylococcus capitis
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<170>SIPOSequenceListing 1.0
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Claims (1)
1. A kit for detecting staphylococcus capitis is characterized by comprising guide RNA of a specific targeting staphylococcus capitis rpoB gene, an RPA amplification primer pair, a hydrated twist Amp basic kit reaction dry ball, LbCas12a protein, a single-stranded DNA probe, an RNase inhibitor and a buffer solution; wherein the nucleotide sequence of the guide RNA of the specific targeting staphylococcus capitis rpoB gene is shown as SEQ No.1, the sequence of the RPA amplification primer pair is shown as SEQ No.2 and SEQ No.3, and the sequence of the single-stranded DNA probe is shown as SEQ No. 6.
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| CN201910370161.2A CN110129463B (en) | 2019-05-06 | 2019-05-06 | Kit for detecting staphylococcus capitis |
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| CN201910370161.2A CN110129463B (en) | 2019-05-06 | 2019-05-06 | Kit for detecting staphylococcus capitis |
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| CN110129463B true CN110129463B (en) | 2020-08-28 |
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| CN112961929B (en) * | 2021-05-18 | 2021-08-10 | 至善时代智能科技(北京)有限公司 | Primer group, kit and method for staphylococcus in species level identification environment |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10253365B1 (en) * | 2017-11-22 | 2019-04-09 | The Regents Of The University Of California | Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10253365B1 (en) * | 2017-11-22 | 2019-04-09 | The Regents Of The University Of California | Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs |
Non-Patent Citations (3)
| Title |
|---|
| Conversion of staphylococcal pathogenicity islands to CRISPR-carrying antibacterial agents that cure infections in mice;Geeta Ram1等;《Nat Biotechnol》;20180924;第36卷(第10期);971-976 * |
| CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity;Janice S. Chen等;《Science》;20180427;第360卷(第6387期);436-439页，补充实验图S1-S13，表S1，共1-24 * |
| rpoB Gene Sequence-Based Identification of Staphylococcus Species;Michel Drancourt等;《JOURNAL OF CLINICAL MICROBIOLOGY》;20020430;第40卷(第4期);1333-1338 * |
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