CN113512598B - Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis and special primer and probe thereof - Google Patents
Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis and special primer and probe thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis, and a special primer and a special probe thereof, belonging to the technical field of biomedical detection. The kit provided by the invention comprises nucleic acid extracting solution, detection solution a, detection solution b and SAT enzyme solution, primers and probes which are more suitable for detection of the bordetella pertussis are optimally designed, and each component is added step by step in the detection process to perform step reaction, so that the quick and accurate detection of the bordetella pertussis can be realized, the specificity is good, the sensitivity is high, and the amplified product RNA is easy to degrade and does not cause sample cross contamination and environmental pollution.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, in particular to a real-time fluorescent nucleic acid isothermal amplification detection kit of pertussis and a special primer and a probe thereof, and particularly relates to a primer, a probe and a related kit used in the real-time fluorescent nucleic acid isothermal amplification detection of the pertussis (bordetella pertussis, BP) combined by a specific target capturing technology and a real-time fluorescent nucleic acid isothermal amplification detection technology (Simultaneous Amplification and Test, SAT).
Background
Pertussis is mainly a severe acute respiratory infectious disease caused by pertussis bacillus, and detection of the pertussis bacillus is not only meaningful for identifying pathogens, but also is meaningful for detecting pathogens in environments possibly polluted by the pathogens, such as river water, sewage, object surface attachments and the like, and samples of drinking water, food and the like.
At present, the pertussis mainly detected by means of PCR of a nasopharyngeal swab and serum pertussis toxin IgG, wherein a PCR detection product is DNA, is not easy to degrade and pollute the environment, has poor repeatability of manual detection, and increases the risk of contact infection.
The real-time fluorescent nucleic acid isothermal amplification detection technology (Simultaneous Amplification and Testing, SAT for short) is a method for directly and rapidly detecting RNA, and is different from the real-time fluorescent PCR for detecting DNA in that: the former detection system has one more reverse transcription step, and nucleic acid amplification is performed at one temperature (42 ℃) and thus thermal cycling is not required. However, the application of SAT technology in the detection of different types of pathogens is subject to different problems, requiring specific design for specific analysis of pathogen characteristics.
The applicant's prior patent document CN111378724a relates to an RNA amplification detection method, and in the examples, an application of the method in detection of bordetella pertussis is disclosed, and the disclosed method requires that a probe a for identifying the RAN to be detected (the 3' end of the probe a is a sequence specifically combined with the RNA to be detected, and the 5' end of the probe a is a first primer sequence), and a section of sequence non-homologous to the RNA to be detected is introduced into the first primer sequence as a common primer, so that the detection sensitivity can reach 100 copies/reaction, but still cannot meet the requirement of higher detection sensitivity.
Disclosure of Invention
In view of one or more of the problems in the prior art, the present invention provides a real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis, comprising:
(1) Nucleic acid extract: comprising a solid support comprising a specific capture probe for capturing a detection sequence and a first primer for specifically binding to a target sequence;
(2) Detection liquid a: comprising a second primer that cooperates with the first primer for amplifying a target sequence;
(3) Detection liquid b: comprising a first primer and a target detection probe, wherein the target detection probe specifically binds to an amplified product RNA copy of the target;
(4) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase;
the nucleotide sequence of the first primer is shown as SEQ ID NO. 3, the nucleotide sequence of the second primer is shown as SEQ ID NO. 4, and the nucleotide sequence of the target detection probe is shown as SEQ ID NO. 5.
The nucleotide sequence of the specific capture probe is shown as SEQ ID NO. 2.
The kit further comprises:
(5) Washing liquid: it contains NaCl and SDS; and/or
(6) Mineral oil; and/or
(7) Positive control: a system comprising a bordetella pertussis nucleic acid; and/or
(8) Negative control: a system that does not contain bordetella nucleic acid.
In the above kit, the nucleic acid extracting solution comprises the following components: 250-800 mM HEPES, 4-10% lithium dodecyl sulfate, 1-50 mu m of the specific capture probe, 50-500 mg/L magnetic beads, 25-150 pmol/mL of the first primer; the components of the detection liquid a are as follows: 10-50mM Tris,5-40mM KCl,10-40mM MgCl 2 1-20mM NTP,0.1-10mM dNTPs,1-10% PVP40, 250-750 pmol/mL of the second primer;the components of the detection liquid b are as follows: 10-50mM Tris,5-40mM KCl,10-40mM MgCl 2 1-20mM NTP,0.1-10mM dNTPs,1-10% PVP40, 143-857 pmol/mL of the first primer, 143-857 pmol/mL of the target detection probe; the SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10 mM of HEPES pH7.5, 10-100 mM of N-acetyl-L-cysteine, 0.04-0.4 mM of zinc acetate, 10-100 mM of trehalose, 40-200 mM of Tris-HCl pH 8.0, 40-200 mM of KCl, 0.01-0.5 mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
In the kit, the components of the washing liquid are as follows: 5-50 mM HEPES, 50-500 mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; the positive control is a dilution of in vitro transcribed pertussis ptxa gene.
In another aspect, the present invention provides a primer and probe combination for real-time fluorescent nucleic acid isothermal amplification detection of bordetella pertussis, for use in the above kit, comprising:
the nucleotide sequence of the target detection probe is shown as a first primer shown as SEQ ID NO. 3, a second primer shown as SEQ ID NO. 4 and a target detection probe shown as SEQ ID NO. 5.
The primer and probe combination also comprises a specific capture probe with a nucleotide sequence shown as SEQ ID NO. 2.
In yet another aspect, the present invention provides a non-disease diagnostic method for detecting a bordetella pertussis nucleic acid, comprising the steps of:
1) Adding a nucleic acid extracting solution into the sample for nucleic acid extraction to obtain an analysis detection sample;
2) Adding detection liquid a into the analysis detection sample to perform a first-step reaction to obtain a first-step reaction liquid;
3) Adding SAT enzyme solution into the first-step reaction solution to perform a second-step reaction to obtain a second-step reaction solution;
4) Adding detection liquid b into the reaction liquid of the second step to perform a third step reaction, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection;
5) Performing result judgment according to the dt value of the real-time fluorescence detection obtained in the step 4);
if dt is less than or equal to 35, the sample contains pertussis nucleic acid; if dt >35, the sample does not contain bordetella pertussis nucleic acid.
In the method, the condition of the first reaction in the step 2) is that the temperature is kept between 40 and 45 ℃ for 3 to 15 minutes; preheating the SAT enzyme solution in the step 3) before use, wherein the preheating temperature is 41-43 ℃; the condition of the second reaction in the step 3) is 41-43 ℃ for 3-15min; the condition of the third reaction in the step 4) is 41-43 ℃ for 30-50min.
The sample sources include river water, sewage, object surface attachments, drinking water, food and aerosol.
The real-time fluorescent nucleic acid isothermal amplification detection kit for the bordetella pertussis based on the technical scheme comprises a nucleic acid extracting solution, a detection solution a, a detection solution b, a SAT enzyme solution and the like, wherein the nucleic acid extracting solution can comprise a specific capture probe for combining a detection sequence and a first primer for combining with a target sequence specifically, the detection solution a can comprise a second primer, the detection solution b can comprise the first primer, the target detection probe and the like, and the SAT enzyme solution comprises RNA polymerase, reverse transcriptase and the like required in the reaction process. According to the invention, the primer and the probe which are more suitable for the detection of the pertussis nucleic acid are obtained according to the optimal design of the pertussis ptxa gene, and the reaction liquid is added step by step to react step by step in the use process, so that the mutual interference between different primers and probes can be avoided, the system reaction is simpler, and the high-sensitivity and high-specificity detection of the pertussis can be realized. The method is not only used for detecting the pertussis bacillus in the medical field, but also used for detecting the pertussis bacillus in non-medical diagnosis samples, such as environmental samples of river water, sewage, object surface attachments and the like, and samples of drinking water, food and the like, and is suitable for being popularized and used in a large range.
Compared with the existing detection method, the method has the following advantages:
(1) The primer and the probe set in the kit are obtained by optimizing design according to the pertussis bacteria ptxa gene, and are different from the prior patent document CN111378724A of the applicant, which is required to introduce a common primer sequence which is non-homologous with the RNA of the sample to be detected into the primer, the primer probe set which is more suitable for the detection of the pertussis bacteria nucleic acid is obtained by directly optimizing design according to the pertussis bacteria ptxa gene, and different components are added step by step in the detection process so as to realize higher sensitivity detection. The results of the examples show that the sensitivity of the kit and the method provided by the invention to the detection of the pertussis can reach 10 copies/reaction, which is one order of magnitude higher than that of the detection of the pertussis (100 copies/reaction) in the prior patent document CN111378724A of the applicant, so that the kit and the method provided by the invention can meet the requirement of higher sensitivity.
(2) The invention adopts a real-time fluorescent nucleic acid isothermal amplification detection technology (Simultaneous Amplification and Testing, SAT for short) to detect the pertussis bacillus, avoids operations such as heating, centrifuging and the like for extracting RNA in the prior art (such as RT-PCR), simplifies experimental steps and reaction systems, is easy to realize automation, greatly improves detection sensitivity, and reduces the risk of infection of operators.
(3) The invention synchronously carries out the amplification and detection of nucleic acid in the same closed system, and the whole process has no temperature rise and fall and circulation, thus greatly shortening the required time and reducing the design and production cost of the PCR instrument.
(4) The amplified product of the invention is RNA, which is easy to degrade in nature, and compared with PCR amplified DNA, the invention has the advantages of easy pollution control, small cross influence and no cross pollution and environmental pollution of samples.
Drawings
FIG. 1 is an amplification curve of group 1 (panel A) and group 2 (panel B) primers and probes of example 1 against a series of concentrations of a Bordetella pertussis positive standard;
FIG. 2 is an amplification curve of a pertussis clinical sample.
Detailed Description
Aiming at the defects of the pertussis detection method in the prior art, the invention detects the pertussis by using a nucleic acid constant temperature synchronous amplification detection method, and provides a nucleic acid constant temperature synchronous amplification detection kit for the pertussis detection, a special primer, a special probe and a detection method thereof.
The present invention will be described in detail with reference to specific embodiments and drawings.
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.
The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular cloning guidelines (Molecular Cloning: A Laboratory Manual) Sambrook, j., russell, david w., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).
The various biomaterials described in the examples were obtained by merely providing a means of experimental acquisition for the purposes of specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
All primers, fluorescent probes and in vitro transcribed RNA products mentioned in the present invention are synthesized using the prior art.
Example 1: design of special primer and probe for detecting ptxa gene of bordetella pertussis by real-time fluorescent nucleic acid isothermal amplification
According to the disclosed pertussis nucleic acid sequence of Genbank database, the inventor selects a sequence which is determined to be highly conserved on one segment of pertussis ptxa gene and has larger difference with other similar pathogens as a detection sequence (the nucleotide sequence of corresponding DNA is shown as SEQ ID NO: 1), and designs primers and probes according to the design principle of primers and probes so as to perform real-time fluorescent nucleic acid isothermal amplification detection on the pertussis.
In this example, a total of sets of primers and probes were designed, wherein the following two sets of primers and probes (set 1 and set 2) were selected for detection verification of a bordetella pertussis positive control (described in detail below) and a negative control (a system containing no bordetella pertussis nucleic acid, such as deionized water), and thus primer and probe sets having good specificity, sensitivity and reproducibility were selected for detection of bordetella pertussis.
Group 1:
specific capture probes:
GGTGATGCCGTTGTGATAGACCCGCGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:2)；
a first primer:
AATTTAATACGACTCACTATAGGGAGAGACGATCGACGCTACGGACCTTCGCG(SEQ ID NO:3)；
and (2) a second primer: TCGCTACGTCAGCCAGCAGACTCG (SEQ ID NO: 4);
target detection probes: CGACCUAUCCCAACCCCUACACACGGAGGUCG (SEQ ID NO: 5), FAM fluorescence labeling at the 5 'end and DABCYL fluorescence labeling at the 3' end;
wherein the amplification regions of the first primer and the second primer in the ptxa gene detection sequence are used as a ptxa gene target sequence, and the target detection probe is specifically combined with the amplified product RNA copy of the target sequence;
group 2:
specific capture probe-1, specific capture probe in the same group 1 (SEQ ID NO: 2);
first primer-1: AATTTAATACGACTCACTATAGGGAGAGCTATGCGCACCAATGTGCC (SEQ ID NO: 6);
second primer-1: ACGACCACGGAGTATTCCAACGCT (SEQ ID NO: 7);
target detection probe-1: CCAGGCCAGCAGACUCGCGCCUGG (SEQ ID NO: 8) which is fluorescently labeled at the 5 'end with FAM and at the 3' end with DABCYL.
The positive control in this example was prepared by the following steps:
(1) Synthesizing a segment of conservative fragment (SEQ ID NO: 1) of the pertussis ptxa gene by a chemical synthesis method, and constructing the segment on a common plasmid vector containing a T7 promoter sequence;
(2) RNA fragments were transcribed using a commercial T7 promoter external transcription kit (sigma), and after purification, RNA copy numbers were calculated by UV as positive controls.
Positive standards obtained by serial dilution of positive control with the above two sets of primers and probes (set 1 and set 2) were each subjected to a reaction (concentration of 10 respectively 4 cobies/reaction, 10 3 cobies/reaction, 10 2 Real-time fluorescent nucleic acid isothermal amplification assays (specific assays are described in example 3 below) were performed on copies/reaction, 10 copies/reaction, and negative controls. As a result, as shown in FIG. 1, wherein A is the real-time fluorescent nucleic acid isothermal amplification detection curve of the primer and probe of group 1, B is the real-time fluorescent nucleic acid isothermal amplification detection curve of the primer and probe of group 2, it is seen that the detection sensitivity of the primer and probe of group 1 can reach 10 copies/reaction, and the detection sensitivity of the primer and probe of group 2 can reach only 10 2 The probes/reactions, and therefore the detection sensitivity of the primers and probes of set 1 was significantly higher than that of set 2, the primers and probes shown in set 1 were selected for real-time fluorescent nucleic acid isothermal amplification detection of bordetella pertussis in the examples below.
Example 2: real-time fluorescent nucleic acid isothermal amplification detection kit for pertussis ptxa gene
The kit for detecting the pertussis nucleic acid provided by the embodiment is a kit for detecting the ptxa gene of the pertussis based on the RNA nucleic acid constant temperature synchronous amplification detection principle, and specifically comprises the following components:
(1) Viral nucleic acid extract: a method for extracting and purifying a bordetella pertussis nucleic acid in a sample comprising 250-800 mM HEPES, 4-10% lithium dodecyl sulfate (LLS), 1-50 μm specific capture probe (SEQ ID NO: 2), 50-500 mg/L magnetic beads, 25-150 pmol/mL first primer (SEQ ID NO: 3);
(2) Detection liquid a: it comprises 10-50mM Tris,5-40mM KCl,10-40mM MgCl 2 1-20mM NTP,0.1-10mM dNTPs,1-10% PVP40, 250-750 pmol/mL of a second primer (SEQ ID NO: 4);
(3) Detection liquid b: it comprises 10-50mM Tris,5-40mM KCl,10-40mM MgCl 2 1-20mM NTP,0.1-10mM dNTPs,1-10% PVP40, 143-857 pmol/mL of first primer (SEQ ID NO: 3), 143-857 pmol/mL of target detection probe (SEQ ID NO: 5);
(4) SAT enzyme solution: it mainly contains 16000-160000U/mL of M-MLV reverse transcriptase and 8000-80000U/mL of RNA polymerase (e.g., T7 RNA polymerase), specifically 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10 mM HEPES pH7.5, 10-100 mM N-acetyl-L-cysteine, 0.04-0.4 mM zincacetate, 10-100 mM trehalose, 40-200 mM Tris-HCl pH 8.0, 40-200 mM KCl, 0.01-0.5 mM EDTA, 0.1-1% (v/v) Triton X-100 and 20-50% (v/v) glycol (glycerol).
For ease of detection, this example provides a kit further comprising the following components:
(5) Washing liquid: the magnetic bead washing agent is used for washing magnetic beads in water phase, and the formula of the magnetic bead washing agent comprises 5-50 mM HEPES, 50-500 mM NaCl, 1% SDS and 1-10mM EDTA;
(6) Mineral oil: mineral oil for washing the magnetic bead organic phase;
(7) A positive control; can be in vitro transcribed from the pertussis ptxa gene (1X 10 6 Dilutions of copies/mL (prepared in example 1);
(8) Negative control: systems that do not contain bordetella nucleic acid, such as deionized water or sample preservation solutions (which contain high concentrations of detergent and physiological saline).
Example 3: method for detecting ptxa gene of bordetella pertussis by real-time fluorescent nucleic acid isothermal amplification
The method of the embodiment detects the ptxa gene of the pertussis based on the RNA constant temperature synchronous amplification detection principle, and uses the kit provided in the embodiment 2 to detect whether the oropharynx swab/sputum sample obtained from the subject contains the pertussis nucleic acid or not, and the specific operation steps are as follows:
3.1 sample preparation
Respectively taking 250 mu L of pertussis positive control (described in example 1), 250 mu L of negative control and 250 mu L of sample to be tested (oropharynx swab sample/sputum sample) and placing the samples in a sample treatment tube for standby;
3.2 nucleic acid extraction
(1) Adding 100 mu L-800 mu L of virus nucleic acid extracting solution (HEPES 500mM, LLS 8%, specific capture probe (SEQ ID NO: 2) 15 mu M, magnetic beads 150mg/L and first primer (SEQ ID NO: 3) concentration 100 pmol/mL) into each sample treatment tube, mixing uniformly, preserving heat at 60 ℃ for 10 minutes, and standing at room temperature for 5-10 minutes;
(2) The sample treatment tube is placed on a magnetic bead separation device and kept stand for 2 to 5 minutes. After the magnetic beads are adsorbed on the tube wall, the sample treatment tube is kept on the magnetic bead separation device, the liquid is sucked and discarded, and the magnetic beads are reserved. Adding 1mL of washing liquid (HEPES 25mM, naCl150mM, 1% SDS, EDTA2.5 mM), shaking uniformly, standing for 2-5 minutes, discarding the liquid, retaining the magnetic beads, then adding 800 mu L of washing liquid and 150 mu L of mineral oil, standing for 2-5 minutes after shaking uniformly, discarding the liquid, and retaining the magnetic beads;
(3) The sample processing tube is removed from the magnetic bead separation device, and the magnetic bead-nucleic acid complex is contained in the tube for standby.
3.3 SAT amplification assay
(1) mu.L of detection solution a (Tris 15mM, mgCl) was added to each sample treatment tube 2 15mM, dNTP 2.5mM, NTP 3mM, PVP 40% and KCl 10mM, the concentration of the second primer (SEQ ID NO: 4) was 500 pmol/mL) and the magnetic beads were resuspended by shaking;
(2) Adding 40 mu L of the reaction detection solution a which is uniformly mixed by vibration into a clean micro-reaction tube, and adding 50 mu L of mineral oil into each reaction tube at 42 ℃ for 5-10min. 25. Mu. LSAT enzyme solution (pre-warmed at 42℃in advance, containing M-MLV reverse transcriptase 60000U/mL, T7 RNA polymerase 40000U/mL, 10mM HEPES pH7.5, 15mM N-acetyl-L-cysteine, 0.15mM zinc acetate, 20mM trehalose, 100mM Tris-HCl pH 8.0, 80mM KCl, 0.25mM EDTA, 0.5% (v/v) Triton X-100 and 30% (v/v) glycerol) was added to the microreactor tube at 42℃for 5-10min;
(3) mu.L of detection solution b (Tris 15mM, mgCl) was added to the micro-reaction tube 2 15mM, dNTP 2.5mM, NTP 3mM, PVP40 1%, KCl 10mM, first primer (SEQ ID NO: 3) at 429pmol/mL, target detection probe (SEQ ID NO: 5) at 429pmmol/mL), the reaction tube is quickly transferred to a constant temperature fluorescence detection instrument, the reaction is carried out for 40 minutes at 42 ℃, and the fluorescence is detected once every 1 minute for 40 times; the fluorescein channel selects for the FAM channel.
3.4, result determination
And setting a threshold line according to a curve obtained by the SAT amplification result, reading the dt value, and judging the result.
Threshold setting: the highest point of the normal negative control amplification curve is just exceeded with the threshold line. dt represents the abscissa reading corresponding to the intersection of the sample curve with the threshold line. The result judgment criteria were:
if the FAM channel dt is less than or equal to 35, the sample is positive, namely the sample contains pertussis nucleic acid;
FAM channel dt >35, the sample is negative, i.e. the sample does not contain pertussis nucleic acid.
The method is used for respectively carrying out real-time fluorescent nucleic acid isothermal amplification detection on 10 pertussis oropharynx swab samples (physiological saline matrix) and 10 pertussis sputum samples. The results are shown in FIG. 2 and Table 1 below, and it can be seen that the test results of 20 clinical samples are positive.
Table 1: real-time fluorescent nucleic acid isothermal amplification detection results of 10 pertussis oropharynx swab samples and 10 pertussis sputum samples
| CT value | CT value | ||
| Swab sample 1 | 24.4 | Sputum sample 1 | 24.3 |
| Swab sample 2 | 17.2 | Sputum sample 2 | 18.6 |
| Swab sample 3 | 24.9 | Sputum sample 3 | 21.5 |
| Swab sample 4 | 21 | Sputum sample 4 | 21.7 |
| Swab sample 5 | 24.3 | Sputum sample 5 | 21.7 |
| Swab sample 6 | 23.9 | Sputum sample 6 | 29.1 |
| Swab sample 7 | 25.8 | Sputum sample 7 | 19 |
| Swab sample 8 | 22.6 | Sputum sample8 | 27.4 |
| Swab sample 9 | 23.3 | Sputum sample 9 | 28.4 |
| Swab sample 10 | 21.6 | Sputum sample 10 | 29 |
With reference to this embodiment, the detection of other clinical diagnostic samples (such as nasal swabs, alveolar relational fluids, blood, feces, etc.) and non-clinical diagnostic samples (such as environmental samples including river water, sewage, object surface attachments, aerosols, etc.), and pertussis in samples including drinking water, food, etc.), all show high detection sensitivity.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (9)
1. A real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis, comprising:
(1) Nucleic acid extract: comprising a solid support comprising a specific capture probe for capturing a detection sequence and a first primer for specifically binding to a target sequence;
(2) Detection liquid a: comprising a second primer that cooperates with the first primer for amplifying a target sequence;
(3) Detection liquid b: comprising a first primer and a target detection probe, wherein the target detection probe specifically binds to an amplified product RNA copy of the target;
(4) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase;
the nucleotide sequence of the first primer is shown as SEQ ID NO. 3, the nucleotide sequence of the second primer is shown as SEQ ID NO. 4, the nucleotide sequence of the target detection probe is shown as SEQ ID NO. 5, and the nucleotide sequence of the specific capture probe is shown as SEQ ID NO. 2.
2. The kit of claim 1, wherein the solid support is a magnetic particle.
3. The kit of claim 1 or 2, wherein the kit further comprises:
(5) Washing liquid: it contains NaCl and SDS; and/or
(6) Mineral oil; and/or
(7) Positive control: a system comprising a bordetella pertussis nucleic acid; and/or
(8) Negative control: a system that does not contain bordetella nucleic acid.
4. The kit according to claim 1 or 2, wherein,
the nucleic acid extracting solution comprises the following components: 250-800 mM HEPES, 4-10% lithium dodecyl sulfate, 1-50 mu m of the specific capture probe, 50-500 mg/L magnetic beads, 25-150 pmol/mL of the first primer;
the components of the detection liquid a are as follows: 10-50mM Tris,5-40mM KCl,10-40mM MgCl 2 1-20mM NTP,0.1-10mM dNTPs,1-10% PVP40, 250-750 pmol/mL of the second primer;
the components of the detection liquid b are as follows: 10-50mM Tris,5-40mM KCl,10-40mM MgCl 2 1-20mM NTP,0.1-10mM dNTPs,1-10% PVP40, 143-857 pmol/mL of the first primer, 143-857 pmol/mL of the target detection probe;
the SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10 mM of HEPES pH7.5, 10-100 mM of N-acetyl-L-cysteine, 0.04-0.4 mM of zinc acetate, 10-100 mM of trehalose, 40-200 mM of Tris-HCl pH 8.0, 40-200 mM of KCl, 0.01-0.5 mM of EDTA, 0.1-1% of Triton X-100 with v/v and 20-50% of glycl.
5. The kit according to claim 3, wherein,
the components of the washing liquid are as follows: 5-50 mM HEPES, 50-500 mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA;
the positive control is a dilution of in vitro transcribed pertussis ptxa gene.
6. A primer and probe combination for real-time fluorescent nucleic acid isothermal amplification detection of bordetella pertussis for use in the kit of any one of claims 1-5, comprising:
a first primer with a nucleotide sequence shown as SEQ ID NO. 3, a second primer with a nucleotide sequence shown as SEQ ID NO. 4, a target detection probe with a nucleotide sequence shown as SEQ ID NO. 5 and a specific capture probe with a nucleotide sequence shown as SEQ ID NO. 2.
7. A non-disease diagnostic method for detecting a bordetella pertussis nucleic acid using the kit of any one of claims 1 to 5, comprising the steps of:
1) Adding a nucleic acid extracting solution into the sample for nucleic acid extraction to obtain an analysis detection sample;
2) Adding detection liquid a into the analysis detection sample to perform a first-step reaction to obtain a first-step reaction liquid;
3) Adding SAT enzyme solution into the first-step reaction solution to perform a second-step reaction to obtain a second-step reaction solution;
4) Adding detection liquid b into the reaction liquid of the second step to perform a third step reaction, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection;
5) Performing result judgment according to the dt value of the real-time fluorescence detection obtained in the step 4);
if dt is less than or equal to 35, the sample contains pertussis nucleic acid; if dt >35, the sample does not contain bordetella pertussis nucleic acid.
8. The method according to claim 7, wherein the conditions of the first reaction in step 2) are 40 to 45 ℃ for 3 to 15 minutes;
preheating the SAT enzyme solution in the step 3) before use, wherein the preheating temperature is 41-43 ℃;
the condition of the second reaction in the step 3) is 41-43 ℃ for 3-15min;
the condition of the third reaction in the step 4) is 41-43 ℃ for 30-50min.
9. The method of claim 7 or 8, wherein the sample source comprises river water, sewage, object surface attachments, drinking water, food, aerosol.
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