CN112301158B - RDA method and kit for rapidly detecting Classical Swine Fever Virus (CSFV) - Google Patents
RDA method and kit for rapidly detecting Classical Swine Fever Virus (CSFV) Download PDFInfo
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Abstract
The invention discloses an RDA method and a kit for rapidly detecting swine fever virus (CSFV), which comprise a specific primer pair and an RDA fluorescent label probe, so as to realize safe, specific, sensitive and simple detection of the CSFV, thereby overcoming the defects of the traditional detection technology. The kit provided by the invention can omit the nucleic acid extraction step, realizes the detection of Classical Swine Fever Virus (CSFV) within 20min under the constant temperature condition, has the specificity of 100%, and is suitable for on-site rapid detection, and compared with the common PCR method, the RDA fluorescence method is used for reaction under the constant temperature without changing temperature and complex instruments. The method and the kit thereof have the characteristics of simple and quick operation, good specificity, high sensitivity, low cost and the like, can provide an effective technical means for the on-site quick detection and screening of the Classical Swine Fever Virus (CSFV), and have wide application prospects.
Description
Technical Field
The invention belongs to the technical field of molecular biology. More particularly, it relates to a primer pair, a probe and a related kit for detecting Classical Swine Fever Virus (CSFV) based on RDA fluorescence detection technology.
Background
The swine fever (CLASSICAL SWINE FEVER, CSF) is a highly contact and lethal infectious disease of pigs, which is mainly characterized by high heat, skin and mucous membrane appearance and is mainly transmitted through respiratory tract and digestive tract, caused by swine fever virus (CLASSICAL SWINE FEVER virus, CSFV), has high transmission speed, high morbidity and mortality, brings great economic loss to the global pig industry, is listed as an animal infectious disease which must be reported by the world animal health organization, is listed as a kind of animal epidemic disease by the agricultural department of China, and is one of the most serious animal epidemic diseases endangering the pig industry in China. In order to effectively prevent and control the disease, there is an urgent need for research efforts to enhance CSFV detection techniques. At present, the detection of swine fever mainly comprises the methods of virus separation and identification, serology and molecular biology. The virus separation and identification operation is complicated, the period is long, and the method is not suitable for detecting a large amount of samples; serological methods cannot be used for early diagnosis of CSFV infection; PCR is prone to cross-contamination and cannot be quantified.
The real-time fluorescent quantitative PCR is a highly sensitive nucleic acid quantitative technology developed on the basis of the PCR technology, integrates the characteristics of sensitivity, rapidness and specificity of the traditional PCR technology and the advantages of high sensitivity and high precision quantification of the spectrum technology, and directly detects the change of fluorescent signals in the PCR process to obtain a quantitative result. The method has the advantages of strong specificity, high sensitivity, good repeatability, accurate quantification, high speed, full-closed reaction and the like, and is widely applied to the aspects of molecular diagnosis, molecular biological research, animal and plant quarantine, food safety detection and the like. The PCR detection needs to rely on a PCR instrument or an expensive real-time quantitative PCR instrument and other various matched equipment, and special PCR laboratories and professional operators are required to be equipped, so that the cost and the application range are limited. With the silent rise of in vitro isothermal amplification of nucleic acids, limitations of conventional amplification techniques have changed, and in the last decade, isothermal nucleic acid amplification techniques, such as LAMP (loop-mediated nucleic acid amplification technique), HDA (helicase-dependent isothermal nucleic acid amplification technique) and the like, have been rapidly developed to amplify nucleic acid molecules under isothermal conditions. The techniques can achieve efficient nucleic acid amplification by only maintaining a constant reaction temperature with a temperature control device, thereby eliminating the dependence on a PCR instrument for precisely controlling temperature changes. If nucleic acid amplification can be achieved at lower temperatures, even at ambient temperature, the nucleic acid amplification technique will be further simplified and a wider range of applications of such techniques will be facilitated.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the existing classical swine fever virus detection technology. The research shows that the Classical Swine Fever Virus (CSFV) RDA fluorescence method detection kit realizes the rapid detection of CSFV, and only needs 20-30min from sample treatment to result completion in the whole process of CSFV detection, thereby greatly shortening the conventional detection time and improving the detection efficiency. The technology can be combined with a portable sample processing technology, is not dependent on laboratory equipment, can be used for detecting on the sampling site, and has important significance for controlling diseases such as classical swine fever virus infection and the like.
The invention aims to provide a probe and a primer pair for detecting classical swine fever virus after optimization.
The nucleotide sequence of the probe is shown as SEQ ID NO. 1 or SEQ ID NO. 2.
Preferably, two schemes are used to design the RDA fluorescent-labeled probe, the first scheme being: the conserved sequence of 25-35bp is selected as a probe sequence, a luminous group is marked at the 5 'end, a quenching group is marked at the 3' end, and any position of 5-10 bases is replaced by tetrahydrofuran residue (THF). The second scheme is as follows: the probe length is 46-52 nucleotides, of which at least 30 are located at the 5 'end of the THF site and at least 15 are located at the 3' end. Through series experimental comparison, the two probe designs are suitable for RDA fluorescence detection methods, and have no obvious difference in detection sensitivity and specificity.
The probe with the nucleotide sequence of SEQ ID NO. 1 is characterized in that a luminous group is marked at the 5 'end, a quenching group is marked at the 3' end, and any position of 5-10 bases is replaced by tetrahydrofuran residue (THF), wherein the specific information is as follows:
CSFV-P1（SEQ ID NO .1）：5’-FAM-GAAGA[THF]TGGCCCCTATGCCGTGCAGACCCA-BHQ1 -3′
The nucleotide sequence of the probe is SEQ ID NO. 2, the 36 th base T marks FAM or other luminescent groups from the 5 'end, the 37 th base is replaced by tetrahydrofuran residue (THF), the 38 th base marks BHQ1 or other quenching groups, and the 3' end is subjected to C3-spacer blocking modification, wherein the specific information is as follows:
CSFV-P2（SEQ ID NO .2）：5’-
GTTAAGGTGCACGCATTGGATGGAAGACTGGCCCC[FAM-dT][THF][BHQ1-dT]GCCGTGCAGACCCA[C3-spacer] -3’
the nucleotide sequences of the primer pair are shown as SEQ ID NO. 3 and SEQ ID NO. 4, the target sequence is shown as SEQ ID NO. 5, and the specific information is as follows:
CSFV-F1（SEQ ID NO .3）： 5’-GCACTGAAGGAGAACACGAGTGCTTGATC -3’；
CSFV-R1（SEQ ID NO .4）： 5’-TAGTTGAAAGTGCAGGAAGTTTTCCTTAC-3’。
The invention further aims at providing a kit for detecting the swine fever virus based on the isothermal amplification technology.
The kit comprises a nucleic acid extraction reagent, a isothermal amplification reaction module, positive control and negative control, and the probe and the primer.
Preferably, the isothermal amplification reaction module is a freeze-dried powder reagent of isothermal amplification reaction mixed reagent.
Preferably, the isothermal amplification reaction mixture is an RPA or recombinase-dependent amplification technique (Recombinase-DEPENDENT AMPLIFICATION, RDA).
Another object of the present invention is to provide a kit for detecting classical swine fever virus based on a recombinase-dependent amplification technique (Recombinase-DEPENDENT AMPLIFICATION, RDA).
The recombinase-dependent amplification technique (Recombinase-DEPENDENT AMPLIFICATION, RDA) is realized by the following technical scheme:
According to the invention, a biological informatics method is utilized to carry out analysis simulation and high-throughput virtual screening on a batch of protein structures, and a large number of biological experiments prove that a new recombinase combination with high stability is finally found. Specifically, the invention develops a novel recombinase composition which is a recombinase KX and an auxiliary protein KY, wherein the nucleotide sequence of the recombinase KX is shown as SEQ ID NO.6, the amino acid sequence of the recombinase KX is shown as SEQ ID NO.7, the nucleotide sequence of the auxiliary protein KY is shown as SEQ ID NO.8, and the amino acid sequence of the recombinase KX is shown as SEQ ID NO. 9.
The recombinase KX can be used for replacing the recombinase UvsX or RecA in the RPA reaction, and the KY protein can be used for replacing UvsY protein in the RPA reaction.
The sequence homology of the recombinase KX with the T4 UvsX protein is 50% (201/395). Based on the recombinase combination, the team develops a novel detection method and detection system of a recombinase-dependent amplification (RDA) technology with high stability and high specificity. The preparation process of the recombinase KX is simple, the yield and the stability are greatly improved, and the mass production cost is low. And the amplification technology based on the recombinase combination development has the advantages of short required primer (18-30 bp), low requirement on the length of a target sequence and wide applicability. Furthermore, the technology has good detection specificity and high sensitivity on the nucleic acid target sequence, can realize high-sensitivity and high-precision rapid molecular detection under the constant temperature condition of 25-42 ℃, has low detection cost, is convenient and quick to operate, and has wide application prospect.
The recombinant enzymes KX and protein KY are derived from ESCHERICHIA PHAGE PHT A phage, ESCHERICHIA PHAGE PHT A belongs to the genus Slopekvirus in the subfamily Myoviridae and TEVENVIRINAE.
The recombinant enzymes KX and protein KY can realize a large amount of soluble expression in escherichia coli.
In particular as an alternative, the preparation method comprises the following steps:
s1, introducing a target gene expression fragment into an expression vector to obtain a recombinant expression vector;
S2, transferring the recombinant expression vector into an expression bacterium to obtain a recombinant engineering bacterium;
S3, carrying out induction culture on the recombinant engineering bacteria, enriching the engineering bacteria, carrying out ultrasonic crushing, and centrifuging to obtain unpurified recombinant enzyme;
s4, purifying the unpurified recombinase through chromatography to obtain the recombinase KX. The purified recombinant enzyme KX does not have the phenomenon of coagulation or precipitation at low temperature.
The target gene expression fragment in the step S1 contains a nucleic acid sequence shown as SEQ ID NO.6, the 5 'end of the target gene expression fragment is provided with a BamHI enzyme cutting site adhesive end, and the 3' end of the target gene expression fragment is provided with a Sall enzyme cutting site adhesive end.
Preferably, the expression vector in step S1 is a pET-28a vector.
Preferably, the expressing bacterium in step S2 is escherichia coli.
The preparation process is simple, the yield and the stability are greatly improved, and the mass production cost is low.
Preferably, the reaction system of the recombinase-dependent amplification technique (RDA) comprises the following reagents: recombinant enzymes KX, KY protein, gp32 protein, strand displacement DNA polymerase, reverse transcriptase, exonuclease, creatine kinase, creatine phosphate, tris-buffer, potassium acetate or sodium acetate, PEG20000 or PEG35000, DTT, dNTPs, dATP, probes, primer pairs, magnesium acetate. Preferably, the reaction system further comprises a detection template, such as a sample DNA or RNA to be detected. Preferably, the reaction system further comprises a detection template, such as a sample DNA or RNA to be detected.
Preferably, the reaction conditions of the reaction system are 25-42 ℃ for 10-60min.
More preferably, the reaction conditions of the reaction system are 39 ℃ for 30min.
The reaction principle of the recombinase-dependent amplification (RDA) reaction system is as follows: (1) reverse transcription of RNA into DNA; (2) A recombinase-primer complex formed by combining recombinase with a specific primer of 18-30bp in a reaction system, and searching a target site in a double-stranded DNA template; (3) After the recombinase-primer complex recognizes the template specific sequence, localization occurs and strand exchange is initiated, and the single-stranded binding protein is then bound to the D-Loop structure formed by the displaced DNA strand; (4) The dATP conformation in a recombinase-primer complex hydrolysis system is changed, the 3 'end of a primer is exposed after the recombinase is dissociated and is recognized by DNA polymerase, and the DNA polymerase starts DNA synthesis at the 3' end of the primer according to a template sequence; (5) The DNA polymerase has a strand displacement function, and the double-helix DNA structure of the template is continuously unwound while the primer is extended, and the DNA synthesis process is continuously carried out; (6) The two primers are amplified to form a complete amplicon; (7) In the reaction system, dATP is hydrolyzed into recombinase to be changed into dATP, and phosphocreatine can transfer the phosphate group of the phosphocreatine into dATP molecules under the catalysis of creatine kinase to form dATP, so that the level of the dATP in the reaction system is recovered. The above process is repeated continuously, and finally, the efficient amplification of the nucleic acid is realized.
A kit for detecting Classical Swine Fever Virus (CSFV) based on a recombinase-dependent amplification technology (RDA) is constructed based on the reaction system, and comprises a nucleic acid extraction reagent, an RDA isothermal amplification reaction module, a positive control and a negative control, and the probe and the primer.
Preferably, the RDA isothermal amplification reaction module is freeze-dried powder of RDA isothermal amplification reaction mixed reagent.
Preferably, the RDA isothermal amplification reaction module comprises recombinase KX 60-600 ng/mu L, KY protein 16-192 ng/mu L, single-stranded binding protein gp32 100-1000 ng/mu L, strand displacement DNA polymerase 3-100 ng/mu L, exonuclease 30-200U, creatine kinase 0.1-0.8 mg/ml, creatine phosphate 25-75 mM, tris buffer 20-100mM, PEG2.5% -10%, potassium acetate or sodium acetate 0-150 mM, dATP 1-5 mM, dNTPs 150-600nM each, DTT 1-12 mM, probe 150nM-600nM, and primer pair 150-600nM.
Preferably, the Tris-buffer is Tris-tricine.
Preferably, the concentration of Tris-tricine is 100mM.
The nucleic acid extraction reagent comprises Buffer A and Buffer B. Buffer A is sample lysate, and contains Tris-HCL Buffer system, naOH, SDS, EDTA, guanidine isothiocyanate, tween80 and triton; buffer B contains Tris Buffer system, potassium chloride and magnesium chloride; the positive control is a target gene plasmid containing Classical Swine Fever Virus (CSFV), and the negative control is an empty vector pUC57 plasmid.
Still another object of the present invention is to provide a method for detecting swine fever virus based on a recombinase-dependent amplification technique.
The detection method comprises the following steps: extracting a sample to be detected, taking nucleic acid of the sample to be detected as a template, carrying out real-time fluorescence RDA reaction in the presence of a primer pair, a probe and RDA freeze-dried powder reagent, buffer A and Buffer B of the swine fever virus, and analyzing the sample to be detected according to a real-time fluorescence RDA amplification curve; the nucleotide sequence of the probe is shown as SEQ ID NO. 1 or SEQ ID NO. 2, wherein the reaction temperature is 25-42 ℃, and the reaction time is more than 10 minutes.
Preferably, the method comprises the following steps:
1) Sample processing
Shaking and mixing 20 μL Buffer A and 5 μL positive control/negative control/sample to be detected (pig oral-nasal secretion/blood/tissue), standing at room temperature for 10-15min;
2) System preparation and detection
Adding 25 mu L of Buffer B, shaking and uniformly mixing, adding 50 mu L of mixed solution into an RDA isothermal amplification reaction module, covering a tube cover, shaking and centrifuging, and immediately detecting; the reaction procedure is: the fluorescent signal is collected every minute after 1 minute and 30 cycles at 39 ℃ and the detection is completed after 30 minutes;
3) Result determination
The result is interpreted based on the Time (Tt) at which the fluorescence value generated by the reaction system reaches the Threshold value.
① Positive control: with typical amplification curves present, tt values <25, are valid results;
② Negative control: no amplification curve appears, or Tt value is more than or equal to 30, which is an effective result;
③ The sample to be tested:
a. If Tt value is less than 25, judging positive;
b. If the Tt value is more than or equal to 30, judging negative;
c. If the Tt value is less than or equal to 25 and less than or equal to 30, judging that the Tt value is suspicious, and repeating detection to confirm; the re-detection result is still that the Tt value is less than or equal to 25 and less than 30, the negative control Tt value should be referred, and if the negative control Tt value is more than or equal to 30, the positive result is judged.
From the above technical solutions, the embodiment of the present invention has the following advantages:
1. The kit provided by the invention can be used for detecting swine fever virus RNA in swine oral-nasal secretions, blood and tissues, has the characteristics of simplicity in operation, rapidness and sensitivity, and provides an effective technical means for rapid detection and screening of swine fever viruses.
2. The kit provided by the invention adopts an RDA isothermal amplification detection method, can realize effective amplification of target genes at 37-42 ℃, does not need temperature change, and does not need complex instruments. The reaction time is short, the reaction can be completed within 20-30min, the specificity is 100%, and the detection sensitivity is 10 copies/. Mu.l.
3. In the RDA method, the recombinase KX protein and KY protein have high specificity to the target sequence in the amplification process, and only the primer sequence and the template sequence are completely complementary to start the amplification, so that the specificity of the amplification is greatly improved, and the high-efficiency constant-temperature nucleic acid amplification without background is realized.
Drawings
FIG. 1 is a graph showing the results of ATP hydrolysis activities of 4 proteins in the recombinase screening of example 1 of the present invention.
FIG. 2 is an agarose gel diagram of a isothermal amplification reaction of 4 proteins in the recombinase screen of example 1 of the invention.
FIG. 3 is a three-dimensional structure of KX protein in example 1 of the present invention.
FIG. 4 is a three-dimensional block diagram of the KY protein heptamer in example 1 of the present invention.
FIG. 5 is a graph showing the results of the RDA fluorescence assay kit according to example 1 of the present invention.
FIG. 6 is a graph showing the sensitivity test results of the RDA fluorescence assay kit according to example 2 of the present invention.
FIG. 7 is a graph showing the results of the specific test of the RDA fluorescence assay kit according to example 3 of the present invention.
FIG. 8 is a graph showing the results of a 37-degree stability test of the RDA fluorescence assay kit of example 4 of the present invention.
FIG. 9 is a graph showing the results of a 37-degree stability test of the RDA fluorescence assay kit of example 4 of the present invention.
FIG. 10 is a graph showing the results of a 37-degree stability test of the RDA fluorescence assay kit of example 4 of the present invention.
FIG. 11 is a graph showing the results of a 37-degree stability test of the RDA fluorescence assay kit of example 4 of the present invention.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. It will be apparent to those skilled in the art that various changes, modifications, substitutions, combinations, and simplifications can be made without departing from the spirit and principles of the invention and these are intended to be equivalent arrangements.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
Unless otherwise indicated, the immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, recombinant DNA, etc., employed by this invention are within the skill of the art. See Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLEC. Mu. M.LAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLEC μm LAR BIOLOGY) (edited by F.M.Ausubel et al, (1987)); the enzyme methods series (METHODS IN ENZYMOLOGY) (academic publishing Co): PCR2 practical methods (PCR 2:A PRACTICAL APPROACH) (M.J. MaxFrson (M.J. MacPherson), B.D. Black (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), harlow (Harlow) and Lane editions (1988) antibodies: laboratory Manual (ANTIBODIES, A LABORATORY MANUAL), animal cell culture (ANIMAL CELL C. Mu.M LTURE) (R.I. Fu Lei Xieni (R.I. Freshney) eds. (1987)).
EXAMPLE 1 RDA fluorescence detection kit for Classical Swine Fever Virus (CSFV)
(1) Acquisition of recombinant enzyme KX and KY proteins
The reported recombinase UvsX has poor stability, is difficult to produce in mass production and store for a long time, and in order to solve the problem, the research and development team finally finds a new recombinase KX and auxiliary protein KY thereof by analyzing and simulating a large quantity of protein structures by using a bioinformatics method.
In this embodiment, the research and development team maps the information of key functional sites in the recombinase structure, such as DNA binding sites, ATP hydrolysis sites, etc., to the three-dimensional protein structure to obtain the information of secondary structure and information of tertiary structure, and constructs a data model for screening the recombinase protein structure by integrating the functional residues, the secondary structure features and the space distance of tertiary structure of the primary structure sequence. Through searching templates matched with the recombinase protein in the primary structure from SwissProt, PDB data, 312 protein sequences are primarily screened out, then secondary structure and tertiary structure comparison are respectively carried out, similarity scores are calculated, ranking is carried out according to the similarity scores, and 15 proteins suspected to have the recombinase activity are simulated and screened out.
The 15 proteins are respectively constructed into recombinant protein expression vectors, and after being respectively expressed and purified, the ability of the recombinant protein expression vectors to hydrolyze ATP is detected, wherein 4 proteins have ATP hydrolysis activity and are KX, X-1, X-2 and X-3 proteins respectively. In the experiment, firefly luciferase ATP bioluminescence detection kit is used, and the experiment is carried out strictly according to the operation of the specification, and the result is shown in FIG. 1.
The method comprises the steps of preparing 4 proteins with ATP hydrolytic activity into a constant-temperature amplification system for amplification reaction, wherein the result is shown in figure 2, N is a negative control, P is a positive control amplified by adding T4UvsX, and 1-4 proteins are KX, X-1, X-2 and X-3 respectively, wherein only KX protein has amplification activity. The KX protein is derived from ESCHERICHIA PHAGE PHT A phage, and the three-dimensional structure diagram is shown in FIG. 3.
In the same way we screened the helper protein KY derived from ESCHERICHIA PHAGE PHT a phage for the recombinase KX, the three-dimensional structure of which is shown in figure 4. Wherein the auxiliary protein KY needs to play an active role in the form of heptamers.
Finally obtaining the recombinase KX for RDA amplification, wherein the nucleotide sequence of the recombinase KX is shown as SEQ ID NO.6, and the amino acid sequence of the recombinase KX is shown as SEQ ID NO. 7; the nucleotide sequence of the recombinase KY is shown as SEQ ID NO.8, and the amino acid sequence is shown as SEQ ID NO. 9.
(2) Swine fever virus detection primer and probe design and screening
The complete gene sequence of classical swine fever virus was searched by NCBI (www.ncbi.nlm.nih.gov), and homology alignment and sequence analysis were performed using Clone manager software and BLAST, from which sequences conserved within the species of the pathogen, interspecies variation were selected as target regions. After comparing the whole genome sequences of various hog cholera viruses and analyzing the homology, finally selecting a conserved E2 gene as a target gene (reference sequence GenBank accession number: MH 880912.1), and designing RDA detection primers and probes by using the target fragment. The DNA plasmid, primer and probe sequence of target gene are synthesized by Shanghai JieRui bioengineering Co.Ltd. The highly conserved sequence of the E2 gene of the swine fever virus is screened as follows:
5’-GCACTGAAGGAGAACACGAGTGCTTGATCGGCAACACCACCGTTAAGGTGCA
CGCATTGGATGGAAGACTGGCCCCTATGCCGTGCAGACCCAAAGAAATCATCTCTAGTGCGGGACCTGTAAGGAAAACTTCCTGCACTTTCAACTA-3'（SEQ ID NO .5）
in the embodiment, the design is carried out by adopting the RDA technology primer design principle, the length of the upstream primer and the downstream primer is 18-30bp, 3 primers of the upstream primer and the downstream primer are respectively designed according to the conserved sequence of the E2 gene of the swine fever virus, and the sequences of the primers are as follows:
the upstream primer CSFV-F1: 5'-GCACTGAAGGAGAACACGAGTGCTTGATC-3' A
The upstream primer CSFV-F2: 5'-ACTGAAGGAGAACACGAGTGCTTGATCGGC-3' A
The upstream primer CSFV-F3:5'-GAGAACACGAGTGCTTGATCGGCAACACCA-3' A
Downstream primer CSFV-R1: 5'-TAGTTGAAAGTGCAGGAAGTTTTCCTTAC-3' A
Downstream primer CSFV-R2: 5'-CCTTACAGGTCCCGCACTAGAGATG-3' A
Downstream primer CSFV-R3:5'-GTGCAGGAAGTTTTCCTTACAGGTCCCGC-3' A
The 3 pairs of primers were paired pairwise to form 9 combinations for optimal primer combination screening.
Combination 1: CSFV-F1 and CSFV-R1; combination 2: CSFV-F1 and CSFV-R2 combination 3: CSFV-F1 and CSFV-R3
Combination 4: CSFV-F2 and CSFV-R1; combination 5: CSFV-F2 and CSFV-R2 combination 6: CSFV-F2 and CSFV-R3
Combination 7: CSFV-F3 and CSFV-R1; combination 8: CSFV-F3 and CSFV-R2 combination 9: CSFV-F3 and CSFV-R3
Through a series of experimental screening and evaluation, it was determined that combination 1 (CSFV-F1 and CSFV-R1) is the optimal primer set, specifically:
CSFV-F1（SEQ ID NO .3）： 5’-GCACTGAAGGAGAACACGAGTGCTTGATC -3’；
CSFV-R1（SEQ ID NO .4）： 5’-TAGTTGAAAGTGCAGGAAGTTTTCCTTAC-3’。
In the RDA fluorescence detection technique, two schemes are used to design the RDA fluorescence labeling probe, the first scheme is as follows: the target region is selected to be a 25-35bp conserved sequence, a 5 '-end is marked with a luminescent group, a 3' -end is marked with a quenching group, any position of 5-10 bases is replaced by tetrahydrofuran residue (THF), the nucleotide sequence is a probe of SEQ ID NO.1 in the embodiment, the 5 '-end is marked with the luminescent group, the 3' -end is marked with the quenching group, any position of 5-position bases is replaced by tetrahydrofuran residue (THF), and the specific information is as follows:
CSFV-P1（SEQ ID NO .1）：
5’-FAM-GAAGA[THF]TGGCCCCTATGCCGTGCAGACCCA-BHQ1 -3′
The second scheme is as follows: the probe length is 46-52 nucleotides, of which at least 30 are located at the 5 'end of the THF site and at least 15 are located at the 3' end. In the probe with the nucleotide sequence of SEQ ID NO. 2, the 5 'end of the probe is marked with FAM or other luminescent groups by a 36 th base T, the 37 th base is replaced by tetrahydrofuran residue (THF), the 38 th base is marked with BHQ1 or other quenching groups, and the 3' end is subjected to C3-spacer blocking modification, wherein the specific information is as follows:
CSFV-P2（SEQ ID NO .2）：
5’-GTTAAGGTGCACGCATTGGATGGAAGACTGGCCCC[FAM-dT][THF]
[BHQ1-dT]GCCGTGCAGACCCA[C3-spacer] -3’
Through series experimental comparison, the two probe designs are both suitable for RDA fluorescence detection methods, and have NO obvious difference in detection sensitivity and specificity, wherein the target conserved sequence required by the first probe design is shorter, the requirement on the nucleic acid sequence is low, and in the subsequent examples of the patent, the first probe CSFV-P1 (SEQ ID NO. 1) is used as a detection probe to prepare an RDA isothermal amplification reaction system.
(3) Establishment of Classical Swine Fever Virus (CSFV) RDA detection method
The patent constructs a kit for detecting Classical Swine Fever Virus (CSFV) based on a recombinase dependent amplification technology (RDA), which comprises a nucleic acid extraction reagent, an RDA isothermal amplification reaction module, a positive control and a negative control, wherein the nucleic acid extraction reagent comprises Buffer A and Buffer B, the Buffer A is sample lysate and contains a Tris-HCL Buffer system, naOH, SDS, EDTA, guanidine isothiocyanate, tween80 and triton, and the Buffer B contains a Tris Buffer system, potassium chloride and magnesium chloride; optimal allocation ratio of a reaction system in the RDA isothermal amplification reaction module is shown in table 1, and the optimal allocation ratio comprises the fluorescent labeled probe and the primer; the positive control is a target gene plasmid containing Classical Swine Fever Virus (CSFV), and the negative control is an empty vector pUC57 plasmid.
Table 1 RDA isothermal amplification reaction module reaction system ratios
| Sequence number | Component (A) | Final concentration |
| 1 | Tris-tricine（PH 7.9） | 100mM |
| 2 | Potassium acetate | 50mM |
| 3 | PEG20000 or PEG35000 | 5% |
| 4 | DTT | 2mM |
| 5 | dNTPs | 200nM each |
| 6 | dATP | 2mM |
| 7 | Reverse transcriptase | 200U |
| 8 | Creatine kinase (CREATINE KINASE) | 0.2mg/ml |
| 9 | Creatine phosphate (Creatine phosphate) | 50mM |
| 10 | Strand-displacing DNA polymerase | 50ng/ul |
| 11 | Gp32 protein | 300 ng/ul |
| 12 | Recombinant enzyme KX | 120 ng/ul |
| 13 | Helper protein KY | 60ng/ul |
| 14 | Exonuclease | 50U |
| 15 | Upstream primer | 500nM |
| 16 | Downstream primer | 500nM |
| 17 | Fluorescent-labeled probe | 300nM |
| 18 | Magnesium acetate | 14mM |
The reaction conditions of the reaction system are as follows: reacting at 25-42 deg.C for 10-60min.
The optimal reaction conditions are as follows: the reaction was carried out at 39℃for 30min.
In this example, 3 samples of swabs or secretions, which were positive for Classical Swine Fever Virus (CSFV) RNA as determined by fluorescent quantitative PCR, were collected and tested using the RDA fluorescence assay kit of this patent.
The specific operation is as follows:
Step one, sample processing. Shaking and mixing 20 μL of Buffer A and 5 μL of positive control/negative control/secretion sample to be detected, and standing at room temperature for 10-15min;
And step two, preparing and detecting the system. Adding 25 mu L of Buffer B, shaking and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence reaction module, covering a tube cover, shaking and centrifuging, and immediately detecting; the reaction procedure is: the fluorescent signal is collected every minute after 1 minute and 30 cycles at 39 ℃ and the detection can be completed after 30 minutes;
And step three, judging the result.
① Positive control: with typical amplification curves present, tt values <25, are valid results;
② Negative control: no amplification curve appears, or Tt value is more than or equal to 30, which is an effective result;
③ The sample to be tested:
a. If Tt value is less than 25, judging positive;
b. If the Tt value is more than or equal to 30, judging negative;
c. If the Tt value is less than or equal to 25 and less than or equal to 30, judging that the Tt value is suspicious, and repeating detection to confirm; the re-detection result is still that the Tt value is less than or equal to 25 and less than 30, the negative control Tt value should be referred, and if the negative control Tt value is more than or equal to 30, the positive result is judged.
The test results are shown in table 2 and fig. 5, and the positive control and the negative control conform to the "① positive control: with typical amplification curves present, tt values <25, are valid results; ② Negative control: no amplification curve appears, or Tt value is more than or equal to 25, and is the content of effective result', the Tt value of each sample is less than 25, and the sample is judged to be positive.
Table 2 establishment of the method for detecting the kit
| Negative control | Positive control | Sample 1 | Sample 2 | Sample 3 | |
| Tt value | - | 8.06 | 11.93 | 24.89 | 20.67 |
The results show that the detection method of the RDA fluorescence detection kit established in the embodiment can detect the Classical Swine Fever Virus (CSFV) RNA in swine secretions.
Example 2 RDA fluorescence detection reagent sensitivity test
The positive control was pUC57-E2 plasmid containing Classical Swine Fever Virus (CSFV) conserved gene, and the negative control was empty vector pUC57 plasmid.
The specific operation is as follows:
firstly, diluting positive control plasmids to 10-4 c, and then diluting the positive control plasmids by 10-time gradient to 10-3 c, 10-2 c and 10-1 c respectively.
And step two, sample processing. Taking 5 mu L of plasmids with each concentration in the step one into an EP tube, simultaneously taking 5 mu L of negative control into another EP tube, respectively adding 20 mu L of Buffer A, shaking and mixing uniformly, and standing at room temperature for 10-15min;
And thirdly, preparing and detecting the system. Adding 25 mu L of Buffer B into each tube, shaking and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence reaction module, covering a tube cover, shaking and centrifuging, and immediately detecting; the reaction procedure is: collecting fluorescence signals every minute after 1 minute and 30 cycles at 39 ℃;
And step four, judging the result. Determination criteria:
① Positive control: with typical amplification curves present, tt values < 25, are valid results;
② Negative control: no amplification curve appears, or Tt value is more than or equal to 25, which is an effective result;
③ The sample to be tested:
a. If Tt value is less than 25, judging positive;
b. If the Tt value is more than or equal to 30, judging negative;
c. If the Tt value is less than or equal to 25 and less than or equal to 30, judging that the Tt value is suspicious, and repeating detection to confirm; the re-detection result is still that the Tt value is less than or equal to 25 and less than 30, the negative control Tt value should be referred, and if the negative control Tt value is more than or equal to 30, the positive result is judged.
The results are shown in Table 3 and FIG. 6. The negative control Tt value is NA, and accords with the content that no amplification curve appears or the Tt value is more than or equal to 25 in the judging standard. 10-4 c, 10-3 c, 10-2 c, 10-1 c have Tt values of < 25min, and according to the result determination criteria, 10-4 c, 10-3 c, 10-2 c, 10-1 c are positive.
That is, the sensitivity of the RDA fluorescence detection kit reaches 10 copies.
TABLE 3 sensitivity test results
| Negative control | 10^4 | 10^3 | 10^2 | 10^1 | |
| Tt value | - | 8.06 | 11.93 | 24.89 | 25.03 |
Example 3 RDA fluorescence detection reagent specificity test
Clinically, 1 case of porcine pseudorabies virus (PRV), 1 case of Porcine Circovirus (PCV), 1 case of Porcine Parvovirus (PPV) and 3 cases of swine fever virus (CFSV) are collected, and 6 total cases of 4 samples which are verified to be positive to corresponding pathogens by fluorescent quantitative PCR are detected, and the specificity of the kit is tested.
The specific operation is as follows:
Step one, sample processing. Taking 5 mu L of each positive sample in an EP tube, simultaneously taking 5 mu L of each positive control and negative control of the kit in a new EP tube, respectively adding 20 mu L of Buffer A, shaking and mixing uniformly, and standing at room temperature for 10-15min;
and thirdly, preparing and detecting the system. Adding 25 mu L of Buffer B into each tube, shaking and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence reaction module, covering a tube cover, shaking and centrifuging, and immediately detecting; the reaction procedure is: 39. 60 seconds at the temperature, 30 cycles, and collecting fluorescent signals every 60 seconds;
And step four, judging the result. Determination criteria:
① Positive control: with typical amplification curves present, tt values < 25, are valid results;
② Negative control: no amplification curve appears, or Tt value is more than or equal to 25, which is an effective result;
③ The sample to be tested:
a. If Tt value is less than 25, judging positive;
b. If the Tt value is more than or equal to 30, judging negative;
c. If the Tt value is less than or equal to 25 and less than or equal to 30, judging that the Tt value is suspicious, and repeating detection to confirm; the re-detection result is still that the Tt value is less than or equal to 25 and less than 30, the negative control Tt value should be referred, and if the negative control Tt value is more than or equal to 30, the positive result is judged.
The results are shown in Table 4 and FIG. 7. Positive control, negative control met "① positive control: with typical amplification curves present, tt values <25, are valid results; ② Negative control: no amplification curve appears, or Tt value is greater than or equal to 25, which is the content of effective results ". The Tt values of the corresponding samples are smaller than 25, and positive is judged; tt is not less than 30 or no signal is detected, and negative is judged.
The results prove that the method and the kit contained in the method have excellent specificity.
TABLE 4 specificity test results
Example 4 RDA stability test of fluorescence detection kit
The liquid reagent needs to be stored at low temperature and can not be repeatedly frozen and thawed. The kit is characterized in that the RDA fluorescence reaction module is dried into a powdery reagent in vacuum, the freeze-dried powdery reagent can be stored at normal temperature, the cost of cold chain transportation and low-temperature storage is saved, and the operation is simpler. The stability of the RDA fluorescence detection kit was verified in this example.
The specific operation is as follows:
Eight-tube containing lyophilized reagents were sealed in aluminum foil bags containing a desiccant and stored in a 37 ℃ incubator. 2 reaction wells were taken for testing at 0 day, 30 day, 90 day, 180 day, respectively.
Step one, sample processing. Taking 5 mu L of positive control and negative control of the kit respectively in an EP tube, adding 20 mu L of Buffer A respectively, shaking and mixing uniformly, and standing at room temperature for 10-15min;
And step two, preparing and detecting the system. Adding 25 mu L of Buffer B into each tube, shaking and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence reaction module, covering a tube cover, shaking and centrifuging, and immediately detecting; the reaction procedure is: 39. 60 seconds at the temperature, 30 cycles, and collecting fluorescent signals every 60 seconds;
And step three, judging the result. Determination criteria:
① Positive control: with typical amplification curves present, tt values < 25, are valid results;
② Negative control: no amplification curve appears, or Tt value is more than or equal to 25, which is an effective result;
③ The sample to be tested:
a. If Tt value is less than 25, judging positive;
b. If the Tt value is more than or equal to 30, judging negative;
c. If the Tt value is less than or equal to 25 and less than or equal to 30, judging that the Tt value is suspicious, and repeating detection to confirm; the re-detection result is still that the Tt value is less than or equal to 25 and less than 30, the negative control Tt value should be referred, and if the negative control Tt value is more than or equal to 30, the positive result is judged.
The results are shown in Table 5 and FIG. 8, FIG. 9, FIG. 10, and FIG. 11. The freeze-dried powder of the reagent of the RDA fluorescence reaction module stored for 0 day, 30 day, 90 day and 180 day is tested, each Tt value is less than 25, and the detection results of the reagent in the kit in the patent after freeze-drying are positive in 0 day, 30 day, 90 day and 180 day according to the result judgment standard. The reagent in the kit can be stably stored for at least 3 months at 37 ℃ after being freeze-dried.
TABLE 5 preservation stability at 37℃
| Day 0 | For 30 days | 90 Days | 180 Days | |
| Negative control | - | - | - | - |
| Positive control | 9.45 | 10.26 | 11.65 | 12.03 |
The above description is merely illustrative of the preferred embodiments of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements or the like falling within the spirit and principles of the present invention should be included in the scope of the present invention.
Claims (5)
1. The kit for detecting the swine fever virus is characterized by comprising a nucleic acid extraction reagent, a isothermal amplification reaction module, a positive control, a negative control, a probe and a primer pair;
The nucleotide sequence of the probe is shown as SEQ ID NO.1, the 5 'end of the probe is marked with a luminous group, the 3' end of the probe is marked with a quenching group, and any position in the 5 th base is replaced by Tetrahydrofuran (THF) residue;
the nucleotide sequences of the primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4, and the target sequence is shown as SEQ ID NO. 5;
the isothermal amplification reaction module comprises a recombinase KX protein with an amino acid sequence shown as SEQ ID NO.7 and a KY protein with an amino acid sequence shown as SEQ ID NO. 9;
the isothermal amplification reaction module is a freeze-dried powder reagent of RDA isothermal amplification reaction mixed reagent.
2. The kit of claim 1, wherein the lyophilized powder reagent of the RDA isothermal amplification reaction mixed reagent comprises 16-192 ng/. Mu.L of recombinant enzyme KX 60-600 ng/. Mu. L, KY protein, 16-1000 ng/. Mu.L of single-stranded binding protein gp, 3-100 ng/. Mu.L of strand displacement DNA polymerase, 30-200U of exonuclease, 0.1-0.8mg/ml of creatine kinase, 25-75mM of creatine phosphate, 20-100 mM of Tris buffer, 2.5-10% PEG, 0-150mM of potassium acetate or sodium acetate, 1-5mM of dATP, 150-600nM of dNTPs, 1-12mM of DTT, 150nM-600nM of the probe, and 150-600nM of the primer pair.
3. The kit of claim 1, wherein the nucleic acid extraction reagent comprises Buffer a and Buffer B; bufferA is sample lysate containing Tris-HCL buffer system, naOH, SDS, EDTA, guanidine isothiocyanate, tween80 and triton; the Buffer B contains a Tris Buffer system, potassium chloride and magnesium chloride; the positive control is a plasmid containing a swine fever virus target gene, and the negative control is an empty vector pUC57 plasmid.
4. Use of a kit according to any one of claims 1 to 3 for the preparation of a product for the detection of classical swine fever virus, comprising the steps of:
Extracting nucleic acid of a sample to be detected, carrying out real-time fluorescence RDA reaction in the presence of a primer pair, a probe and RDA freeze-dried powder reagent, bufferA and Buffer B of swine fever virus by taking the nucleic acid of the sample to be detected as a template, and analyzing the sample to be detected according to a real-time fluorescence RDA amplification curve; wherein the nucleotide sequence of the probe is shown as SEQ ID NO.1; wherein the reaction temperature is 25-42 ℃ and the reaction time is more than 10 minutes.
5. The assay of claim 4 wherein the reaction temperature is 39 ℃ and the reaction time is 40 minutes.
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| CN110358867A (en) * | 2019-08-21 | 2019-10-22 | 郑州中道生物技术有限公司 | African hog cholera virus fluorescent type RAA detection kit |
| CN110699490A (en) * | 2019-11-12 | 2020-01-17 | 南宁众册生物科技有限公司 | RAA constant-temperature fluorescence detection primer probe set, kit and method for African swine fever virus CD2V gene |
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| CN110358867A (en) * | 2019-08-21 | 2019-10-22 | 郑州中道生物技术有限公司 | African hog cholera virus fluorescent type RAA detection kit |
| CN110699490A (en) * | 2019-11-12 | 2020-01-17 | 南宁众册生物科技有限公司 | RAA constant-temperature fluorescence detection primer probe set, kit and method for African swine fever virus CD2V gene |
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