CN112301155A - RDA method and kit for rapidly detecting rotavirus - Google Patents
RDA method and kit for rapidly detecting rotavirus Download PDFInfo
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- CN112301155A CN112301155A CN202010081193.3A CN202010081193A CN112301155A CN 112301155 A CN112301155 A CN 112301155A CN 202010081193 A CN202010081193 A CN 202010081193A CN 112301155 A CN112301155 A CN 112301155A
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- rotavirus
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Abstract
The invention discloses an RDA method and a kit for rapidly detecting rotavirus, which comprise a specific primer pair and an RDA fluorescence labeling probe, so as to realize the safe, specific, sensitive and simple detection of Rotavirus (RV), thereby making up the defects of the existing traditional detection technology. The kit provided by the method can save the step of nucleic acid extraction, can realize the detection of rotavirus within 20min at the constant temperature of 37-42 ℃, has the specificity of 100 percent, and is very suitable for on-site rapid detection. Compared with the common PCR method, the RDA fluorescence method is a reaction at constant temperature, does not need temperature change, does not need complex instruments, and has short reaction time. Therefore, the method and the kit thereof have the characteristics of simple and rapid operation, good specificity, high sensitivity, low cost and the like, provide an effective technical means for the on-site rapid detection and screening of rotavirus, and have wide application prospects.
Description
Technical Field
The invention belongs to the technical field of molecular biology. More particularly, relates to a primer pair, a probe and a related kit for detecting rotavirus nucleic acid based on an RDA fluorescence detection technology.
Background
Rotavirus (RV for short) is a double-stranded ribonucleic acid virus, belonging to the reoviridae family. There are seven rotaviruses, which are numbered A, B, C, D, E, F and G in English letters. Among them, Rotavirus (RV), which causes diarrhea in humans, is only A, B, C group, and group a is the most common one. Group a rotavirus is one of the major pathogens causing infantile diarrhea, and it mainly infects small intestinal epithelial cells, causing cell damage, causing diarrhea. Rotavirus is epidemic in summer, autumn and winter every year, the infection route is a feces-oral route, rotavirus infection is from asymptomatic, slight morbidity to serious morbidity, and fatal gastroenteritis, dehydration and electrolyte balance occur in serious cases. The clinical manifestations of acute gastroenteritis include fever, vomiting, abdominal pain and bloodless watery diarrhea, and the symptoms last for 3-9 days. Almost every child about five years of age in the world has been infected with rotavirus at least once.
At present, the classical detection methods for rotavirus diseases in China are electron microscope technology, enzyme-linked immunosorbent assay (ELISA), colloidal gold method and the like. Methods such as electron microscopy, enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR) and the like require special equipment and professional techniques, but the conditions are often difficult to obtain in basic community diagnosis and treatment institutions, and early discovery and prevention of rotavirus are affected. At present, a simple, cheap and high-automation rotavirus detection kit suitable for basic level detection is still lacked.
With the silent rise of in vitro isothermal amplification of nucleic acid, the limitations of the traditional amplification technology are changed, and in the past decade, isothermal nucleic acid amplification technologies, such as LAMP (loop-mediated nucleic acid amplification technology), HDA (helicase dependent isothermal nucleic acid amplification technology), etc., which make the in vitro amplification of nucleic acid simpler and more convenient, have been rapidly developed to amplify viral nucleic acid under isothermal conditions. These techniques can achieve efficient nucleic acid amplification by only requiring a temperature control device to maintain a constant reaction temperature, thereby getting rid of the dependence on a PCR instrument that precisely controls temperature variations. If nucleic acid amplification can be achieved at room temperature, the nucleic acid amplification technique will be further simplified, and the technique will be useful for a wider range of applications. The timely and accurate discovery of infectious disease epidemic situation has important significance for the prevention and treatment of diseases and the control of epidemic situation, so that the research and development of a kit which can be used in basic level diagnosis and treatment institutions and can rapidly detect nucleic acid at normal temperature is very necessary.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing rotavirus detection technology. The research obtains a Rotavirus (RV) RDA fluorescence detection kit, realizes the rapid detection of rotavirus, and only needs 20-30min from sample treatment to result completion in the whole process of RV detection, thereby greatly shortening the conventional detection time and improving the detection efficiency.
The invention aims to provide a probe and a primer pair for detecting rotavirus after optimization.
The nucleotide sequence of the probe is shown as SEQ ID NO.1 or SEQ ID NO. 2.
Preferably, two protocols are used to design RDA fluorescently labeled probes, the first protocol being: selecting 25-35bp conserved sequence as probe sequence in the target region, marking luminous group at 5 'end, marking quenching group at 3' end, and replacing tetrahydrofuran residue (THF) at any position of 5-10 th base. The second scheme is as follows: the probe is 46-52 nucleotides in length, at least 30 of which are located at the 5 'end of the THF site, and at least 15 of which are located at the 3' end. Through serial experimental comparison, the two probe design schemes are both suitable for the RDA fluorescence detection method, and have no obvious difference in detection sensitivity and specificity.
The nucleotide sequence is a probe of 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, any position of the 5 th to 10 th bases is replaced by a tetrahydrofuran residue (THF), and the specific information is as follows:
RV-P1(SEQ ID NO.1)：
5’-FAM-CAACC[THF]CAGCTCTTGAGAATATGGGAATAG-BHQ1-3′
the nucleotide sequence is a probe of SEQ ID NO.2, the 30 th base T at the 5 'end of the probe is marked with FAM or other luminescent groups, the 32 th base is replaced by tetrahydrofuran residue (THF), the 33 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:
RV-P2(SEQ ID NO.2)：
5-GAAGCTGCAGTTGTAGCTGCAACCTCAGC[FAM-dT]C[THF][BHQ1-dT]GAGA ATATGGGAATAG[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:
RV-F1(SEQ ID NO.3)：5’-TGGAGTCTACGCAACAGATGGCCGTC-3’；
RV-R1(SEQ ID NO.4)：5’-ACTCTAGAATATATATCCTGATAATCA-3’。
the invention also aims to provide a reagent kit for detecting rotavirus based on the isothermal amplification technology.
The kit comprises a nucleic acid extraction reagent, a constant temperature amplification reaction module, a positive control, a negative control, the probe and the primer.
Preferably, the isothermal amplification reaction module is a lyophilized powder reagent of an isothermal amplification reaction mixed reagent.
Preferably, the isothermal amplification reaction mixed reagent is an RPA or Recombinase-dependent amplification (RDA) isothermal amplification reaction mixed reagent.
Another objective of the present invention is to provide a reagent kit for detecting rotavirus based on Recombinase-dependent amplification (RDA).
The Recombinase-dependent amplification (RDA) is realized by the following technical scheme:
the invention utilizes a bioinformatics method to analyze and simulate batch protein structures and virtually screen the protein structures at high flux, and finally finds out a new recombinase combination with high stability through a large number of biological experiments. Specifically, the invention develops a novel recombinase combination comprising 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 auxiliary protein KY is shown as SEQ ID No. 9.
The recombinase KX can replace the recombinase UvsX or RecA in the RPA reaction, and the KY protein can replace the UvsY protein in the RPA reaction.
The recombinase KX has 50% of sequence homology with the T4UvsX protein (201/395). Based on the recombinase combination, the team develops a new detection method and a detection system of a recombinase-dependent amplification technology (RDA) with high stability and strong 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 based on the amplification technology developed by the recombinase combination, the required primer is short (18-30bp), the requirement on the length of the target sequence is low, and the applicability is wide. 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 and convenient and rapid operation, and has wide application prospect.
The recombinase KX and the protein KY are derived from Escherichia phase phT4A phage, and Escherichia phase phT4A belongs to the genus Slopekvarus in Myoviridae subfamily.
The recombinase KX and the protein KY can realize a large amount of soluble expression in escherichia coli.
Specifically, as an alternative, the preparation method is as follows:
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, and carrying out enrichment and ultrasonic crushing on the engineering bacteria and then centrifuging to obtain an unpurified recombinase;
s4, carrying out chromatography purification on the unpurified recombinase to obtain the recombinase KX. The purified recombinant enzyme KX does not have the phenomenon of coagulation or precipitation at low temperature.
Wherein, 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 has a BamHI enzyme cutting site cohesive end, and the 3' end of the target gene expression fragment has a Sall enzyme cutting site cohesive end.
Preferably, the expression vector in step S1 is a pET-28a vector.
Preferably, the expression bacteria in step S2 are 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: preferably, the reaction system of the recombinase-dependent amplification technique (RDA) comprises the following reagents: recombinase 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, probe, primer pair and magnesium acetate. Preferably, the reaction system further comprises a detection template, such as DNA or RNA of a sample to be detected.
Preferably, the reaction condition of the reaction system is that the reaction is carried out for 10-60min at 25-42 ℃.
More preferably, the reaction conditions of the reaction system are 39 ℃ for 30 min.
The reaction principle of the recombinase-dependent amplification technology (RDA) reaction system is as follows: (1) reverse transcription of RNA into DNA; (2) recombinase-primer complexes formed by combining recombinase with specific primers of 18-30bp are used for searching target sites in a double-stranded DNA template; (3) after the recombinase-primer complex recognizes a template specific sequence, positioning occurs and strand exchange is initiated, and the single-strand binding protein is combined with a D-Loop structure formed by the displaced DNA strands; (4) the dATP conformation in the recombinase-primer complex hydrolysis system is changed, the 3 'end of the primer is exposed and recognized by DNA polymerase after the recombinase is dissociated, 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, continues to unwind the double-helix DNA structure of the template while the primer is extended, and the DNA synthesis process continues; (6) completing the amplification of the two primers to form a complete amplicon; (7) in the reaction system, dATP is hydrolyzed to supply energy to recombinase and then becomes dADP, phosphocreatine can transfer the phosphate group of the phosphocreatine into a dADP molecule under the catalysis of creatine kinase to form dATP, and therefore the level of dATP in the reaction system is restored. The above process is repeated continuously, and finally the high-efficiency amplification of nucleic acid is realized.
A reagent kit for detecting Rotavirus (RV) based on a recombinase dependent amplification technology (RDA) is constructed based on the reaction system, and comprises a nucleic acid extraction reagent, an RDA constant temperature amplification reaction module, a positive control, a negative control, the probe and the primer.
Preferably, the RDA constant temperature amplification reaction module is freeze-dried powder of a mixed reagent of the RDA constant temperature amplification reaction.
Preferably, the RDA constant temperature amplification reaction module comprises recombinase KX 60-600 ng/mu L, KY protein 16-192 ng/mu L, single-chain binding protein gp 32100-1000 ng/mu L, strand displacement DNA polymerase 3-100 ng/mu L, exonuclease 30-200U, creatine kinase 0.1-0.8mg/ml, phosphocreatine 25-75mM, reverse transcriptase 200U, Tris buffer 20-100mM, PEG 2.5-10%, potassium acetate or sodium acetate 0-150mM, dATP 1-5mM, dNTPs 150-600nM each, DTT 1-12mM, probe 150nM-600nM, and primer pair 150-600 nM.
Preferably, the Tris-buffer is Tris-tricine.
Preferably, the concentration of Tris-tricine is 100 mM.
The nucleic acid extraction reagent comprises Buffer A and Buffer B. Buffer A is sample lysate, containing Tris-HCL Buffer system, NaOH, SDS, EDTA, guanidinium isothiocyanate, Tween80, and Triton; buffer B contains a Tris Buffer system, potassium chloride and magnesium chloride; the positive control is a target gene plasmid containing Rotavirus (RV), and the negative control is an empty vector pUC57 plasmid.
Still another objective of the present invention is to provide a detection method for detecting rotavirus based on recombinase-dependent amplification technology.
The detection method comprises the following steps: extracting nucleic acid of a sample to be detected, performing real-time fluorescence RDA reaction in the presence of a primer pair of rotavirus, a probe, an RDA freeze-dried powder reagent, Buffer A and Buffer B 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 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 uniformly mixing 20 mu L of Buffer A and 5 mu L of positive control/negative control/feces sample to be detected, and standing for 10-15min at room temperature;
2) system formulation and assay
Adding 25 mu L of Buffer B, shaking and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence method reaction module, covering a tube cover, shaking and centrifuging, and immediately detecting; the reaction procedure is as follows: collecting fluorescence signals every minute at 39 ℃ for 1 minute for 30 cycles, and completing detection in 30 min;
3) determination of results
And (4) judging the result according to the Time (Time Threshold, Tt) when the fluorescence value generated by the reaction system reaches a Threshold value.
(ii) positive control: typical amplification curves appear, and the Tt value is less than 25min, which is a valid result;
negative control: no amplification curve appears, or the Tt value is more than or equal to 25min, which is an effective result;
thirdly, the detected sample:
a. if the Tt value is less than 25min, judging the test result to be positive;
b. if the Tt value is more than or equal to 30min, judging the result to be negative;
c. if Tt value is less than or equal to 25min and less than 30min, the result is judged to be suspicious and needs to be repeatedly detected for confirmation; the result of the secondary detection is that the Tt value is more than or equal to 25min and less than 30min, the negative control Tt value is referred to, and if the negative control Tt value is more than or equal to 30min, the positive result is judged.
The invention has the following beneficial effects:
1. the kit provided by the invention is used for detecting rotavirus RNA in a stool sample, has the characteristics of simple operation, rapidness and sensitivity, provides an effective technical means for on-site rapid detection and screening of rotavirus, and has important significance in clinical detection of rotavirus.
2. The kit provided by the invention adopts an RDA constant temperature amplification detection method, can realize effective amplification of the target gene under the condition of 37-42 ℃, and does not need temperature change and 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, recombinase KX protein and KY protein have high specificity to a target sequence in the amplification process, and amplification is started only when a primer is completely complementary with a template sequence, so that the amplification specificity is greatly improved, and high-efficiency constant-temperature nucleic acid amplification without background is realized.
Drawings
FIG. 1 is a graph showing the results of ATP hydrolysis activity of 4 proteins in recombinase screening of example 1 of the present invention.
FIG. 2 is an agarose gel image of the isothermal amplification reaction of 4 proteins in recombinase screening of example 1 of the invention.
FIG. 3 is a three-dimensional structural diagram of KX protein in example 1 of the present invention.
FIG. 4 is a three-dimensional structure diagram of a 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 results of the sensitivity test of the RDA fluorescence assay kit of example 2 of the present invention.
FIG. 7 is a graph showing the results of the 37-degree stability test of the RDA fluorescence assay kit in example 4 of the present invention.
FIG. 8 is a graph showing the results of the 37-degree stability test of the RDA fluorescence assay kit in example 4 of the present invention.
FIG. 9 is a graph showing the results of the 37-degree stability test of the RDA fluorescence assay kit in example 4 of the present invention.
FIG. 10 is a graph showing the results of the 37-degree stability test of the RDA fluorescence assay kit in example 4 of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. It will be appreciated by those skilled in the art that various other changes, modifications, substitutions, combinations, and omissions may be made in the form and detail of the invention without departing from the spirit and scope of the invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Unless otherwise indicated, the present invention employs immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, recombinant DNA and the like, which are within the ordinary skill of the art. See Sambrook (Sambrook), friech (Fritsch) and mani-tius (manitis), molecular cloning: a LABORATORY Manual (MOLEC M LAR CLONING: A LABORATORY MANUAL), 2 nd edition (1989); a Current Manual of molecular BIOLOGY experiments (Current Protocols IN MOLEC. mu.M.LAR BIOLOGY) (edited by F.M. Otsubel (F.M. Ausubel), et al, (1987)); METHODS IN ENZYMOLOGY (METHODS IN Enzymology) series (academic Press): PCR2 practical methods (PCR 2: A PRARVICAL APPROACH) (m.j. macpherson, b.d. heims (b.d. hames) and g.r. taylor (g.r.taylor) editions (1995)), Harlow (Harlow) and raney (Lane) editions (1988) antibodies: a LABORATORY Manual (ANTIBODIES, A Laboratory Manual), and animal cell culture (ANIMAL CELL C μm LTURE) (edited by R.I. Fresheni (R.I. Freshney) (1987)).
Example 1 detection kit for Rotavirus (RV) RDA fluorescence method
(1) Acquisition of recombinase KX and KY proteins
The reported recombinase UvsX has poor stability and is difficult to be produced in quantity and stored for a long time, and in order to solve the problem, a new recombinase KX and an auxiliary protein KY thereof are finally found by analyzing and simulating large batch protein structures by using a bioinformatics method by the research and development team.
In this embodiment, a research and development team extracts key functional site information in a recombinase structure, such as a DNA binding site, an ATP hydrolysis site, and the like, maps the information to a three-dimensional spatial structure of a protein, obtains secondary structure information and tertiary structure information, and constructs a data model for recombinase protein structure screening by integrating functional residues of a primary structure sequence, secondary structure characteristics, and a tertiary structure spatial distance. A template matched with recombinase proteins in a primary structure is searched from SwissProt and PDB data, 312 protein sequences are preliminarily screened, then secondary structure and tertiary structure comparison is respectively carried out, similarity scores are calculated, and 15 proteins suspected to have recombinase activity are screened in a simulated mode according to the similarity score ranking.
The 15 proteins are respectively constructed into recombinant protein expression vectors, after expression and purification, the ATP hydrolysis capacity of the recombinant protein expression vectors is detected, wherein 4 proteins have ATP hydrolysis activity and are KX, X-1, X-2 and X-3 proteins respectively. The firefly luciferase ATP bioluminescence detection kit is used in the experiment, and the experiment is carried out according to the operation of the instruction, and the result is shown in figure 1.
4 proteins with ATP hydrolytic activity are prepared into a constant-temperature amplification system for amplification reaction, the result is shown in figure 2, N is negative control, P is positive control added with T4UvsX for amplification, 1-4 proteins added respectively are KX, X-1, X-2 and X-3, wherein only the KX protein has amplification activity. The KX protein is derived from Escherichia phase phT4A phage and its three-dimensional structure diagram is shown in FIG. 3.
In the same way, we screened the recombinase KX from the helper protein KY of Escherichia phase phT4A phage, and the three-dimensional structure diagram is shown in FIG. 4. Wherein the auxiliary protein KY needs to play an active role in the form of heptamer.
Finally obtaining 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; recombinase KY, the nucleotide sequence of which is shown as SEQ ID NO.8, and the amino acid sequence of which is shown as SEQ ID NO. 9.
(2) Rotavirus detection primer and probe design and screening
Sequences conserved within species, interspecies variation in the present pathogen were selected as target regions by searching for rotavirus whole gene sequences by NCBI (www.ncbi.nlm.nih.gov), homology alignment and sequence analysis using Clone manager software and BLAST. After the whole genome sequence alignment and homology analysis of various rotaviruses, finally, a conserved NSP3 gene is selected as a target gene (reference sequence GenBank accession number: MH769716.1), and RDA detection primer and probe design is carried out on the target segment. And (3) entrusting Shanghai Czeri bioengineering Limited company to synthesize a target gene DNA plasmid, a primer and a probe sequence. The highly conserved sequence of the rotavirus NSP3 gene is selected as follows:
SEQ ID NO.5：
ATGCTCAAGATGGAGTCTACGCAACAGATGGCCGTCTCAATTATTAACTCTTC TTTTGAAGCTGCAGTTGTAGCTGCAACCTCAGCTCTTGAGAATATGGGAATAGAAT ATGATTATCAGGATATATATTCTAGAGTAAAGAATAAATTTGATTTTGTGATGGACGA TTCTGGTGTTAAAAATAATCTGATTGGTAAAGCAATAACTATTGATCAAGCTTTGAA TAATAAATTTGGATCTGCTATAAGAAATAGAAACTGGCTTGCTGATACTTCTAGAGC AGCTAAATTGGATGAGGATGTAAACAAACTAAGAATGATGTTATCATCAAAAGGAA TTGATCAAAAAATGAGAGTTTTAAACGCATGCTTCAGTGTAAAAAGAATACCTGG AAAATCATCATCTATTATTAAATGCACAAAATTGATGCGTGATAAATTGGAACGTGG TGAAGTTGAAGTGGATGATTCATTTGTGGATGAAAAAATGGAAGTGGATACCATTG ACTGGAAATCGCGCTATGAGCAATTGGAGCAAAGGTTTGAATCATTGAAATCCAG GGTAAATGAAAAATATAATAATTG
in the embodiment, the RDA technology primer design principle is adopted for design, the lengths of the upstream primer and the downstream primer are 18-30bp, 3 upstream primers and 3 downstream primers are designed according to the conserved sequence of rotavirus NSP3 gene, and the primer sequences are as follows:
the upstream primer RV-F1: 5'-TGGAGTCTACGCAACAGATGGCCGTC-3'
The upstream primer RV-F2: 5'-ATGCTCAAGATGGAGTCTACGCAACAGA-3'
The upstream primer RV-F3: 5'-CTACGCAACAGATGGCCGTCTCAATTA-3'
The downstream primer RV-R1: 5'-ACTCTAGAATATATATCCTGATAATCA-3'
The downstream primer RV-R2: 5'-TCATTCTTAGTTTGTTTACATCCTCATCC-3'
The downstream primer RV-R3: 5'-GATCAATAGTTATTGCTTTACCAATCAG-3'
And 3 pairs of primers are paired pairwise to form 9 combinations for optimal primer combination screening.
Combination 1: RV-F1 and RV-R1; and (3) combination 2: RV-F1 and RV-R2 combination 3: RV-F1 and RV-R3
And (4) combination: RV-F2 and RV-R1; and (3) combination 5: RV-F2 and RV-R2 combination 6: RV-F2 and RV-R3
And (3) combination 7: RV-F3 and RV-R1; and (4) combination 8: RV-F3 and RV-R2 combination 9: RV-F3 and RV-R3
Through a series of experimental screening and evaluation, combination 1(RV-F1 and RV-R1) is determined as an optimal primer group, and specifically comprises the following steps:
RV-F1(SEQ ID NO.3)：5’-TGGAGTCTACGCAACAGATGGCCGTC-3’；
RV-R1(SEQ ID NO.4)：5’-ACTCTAGAATATATATCCTGATAATCA-3’。
in the RDA fluorescence detection technology, two schemes are adopted to design RDA fluorescence labeling probes, wherein the first scheme is as follows: selecting a conserved sequence of 25-35bp as a probe sequence in a target region, wherein the conserved sequence is a probe sequence, a luminescent group is marked at the 5 'end, a quenching group is marked at the 3' end, and any position in the 5 th-10 th bases is replaced by a tetrahydrofuran residue (THF), the nucleotide sequence is the probe of SEQ ID NO.1, the luminescent group is marked at the 5 'end, the quenching group is marked at the 3' end, and any position in the 5 th bases is replaced by the tetrahydrofuran residue (THF), and the specific information is as follows:
RV-P1(SEQ ID NO.1)：
5’-FAM-CAACC[THF]CAGCTCTTGAGAATATGGGAATAG-BHQ1-3′
the second scheme is as follows: the probe is 46-52 nucleotides in length, at least 30 of which are located at the 5 'end of the THF site, and at least 15 of which are located at the 3' end. In the probe with the nucleotide sequence of SEQ ID NO.2, the 30 th base T at the 5 'end is labeled with FAM or other luminescent groups, the 32 th base is replaced by Tetrahydrofuran (THF), the 33 th base is labeled with BHQ1 or other quenching groups, and the 3' end is subjected to C3-spacer blocking modification, and the specific information is as follows:
RV-P2(SEQ ID NO.2)：
5-GAAGCTGCAGTTGTAGCTGCAACCTCAGC[FAM-dT]C[THF][BHQ1-dT]GAGA ATATGGGAATAG[C3-spacer]-3’
through series of experimental comparisons, the two probe design schemes are both suitable for the RDA fluorescence detection method, and have no obvious difference in detection sensitivity and specificity, wherein a target conserved sequence required by the first probe design is shorter, and the requirement on a nucleic acid sequence is low, and in the subsequent embodiment of the patent, the first probe RV-P1(SEQ ID No.1) is used as a detection probe to prepare an RDA constant temperature amplification reaction system.
(3) Establishment of Rotavirus (RV) RDA detection method
The patent constructs a reagent kit for detecting Rotavirus (RV) based on a recombinase dependent amplification technology (RDA), which comprises a nucleic acid extraction reagent, an RDA constant temperature 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 a sample lysate and contains a Tris-HCL Buffer system, NaOH, SDS, EDTA, guanidinium isothiocyanate, Tween80 and triton, and the Buffer B contains a Tris Buffer system, potassium chloride and magnesium chloride; the optimal proportion of the reaction system in the RDA isothermal amplification reaction module is shown in Table 1, and the reaction system comprises the fluorescence labeling probe and the primer described in the embodiment; the positive control is a target gene plasmid containing Rotavirus (RV), and the negative control is an empty vector pUC57 plasmid.
TABLE 1 RDA constant temperature amplification reaction module reaction system ratio
| Serial number | Components | Concentration of |
|
| 1 | Tris-tricine(PH 7.9) | |
|
| 2 | | 50mM | |
| 3 | PEG20000 or PEG35000 | 5％ | |
| 4 | Dithiothreitol (DTT) | |
|
| 5 | dNTPs | 200nM each | |
| 6 | dATP | 2mM | |
| 7 | Creatine kinase (Creatine kinase) | 0.2mg/ml | |
| 8 | Creatine phosphate (Creatine phosphate) | 50mM | |
| 9 | | 200U | |
| 10 | Strand-displacing DNA polymerase | 50ng/ul | |
| 11 | gp32 protein | 300ng/ul | |
| 12 | Recombinase KX | 120ng/ul | |
| 13 | Accessory protein KY | 60ng/ul | |
| 14 | | 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 for 10-60min at 25-42 ℃.
The optimal reaction conditions are as follows: the reaction was carried out at 37 ℃ for 30 min.
In the embodiment, 3 collected excrement samples which are all positive to rotavirus RNA through fluorescent quantitative PCR verification are tested by using the RDA fluorescent method detection kit.
The specific operation is as follows:
step one, sample processing. Shaking and uniformly mixing 20 mu L of Buffer A and 5 mu L of positive control/negative control/sample to be detected, and standing for 10-15min at room temperature;
step two, 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 fluorescence method reaction module, covering a tube cover, shaking and centrifuging, and immediately detecting; the reaction procedure is as follows: collecting fluorescence signals every minute at 39 ℃ for 1 minute for 30 cycles, and completing detection within 30 min;
and step three, judging a result.
(ii) positive control: typical amplification curves appear, and the Tt value is less than 25min, which is a valid result;
negative control: no amplification curve appears, or the Tt value is more than or equal to 25min, which is an effective result;
thirdly, the detected sample:
a. if the Tt value is less than 25min, judging the test result to be positive;
b. if the Tt value is more than or equal to 30min, judging the result to be negative;
c. if Tt value is less than or equal to 25min and less than 30min, the result is judged to be suspicious and needs to be repeatedly detected for confirmation; the result of the secondary detection is that the Tt value is more than or equal to 25min and less than 30min, the negative control Tt value is referred to, and if the negative control Tt value is more than or equal to 30min, the positive result is judged.
The detection results are shown in table 2 and fig. 5, and the positive control and the negative control are in accordance with the "first positive control: typical amplification curves appear, and the Tt value is less than 25min, which is a valid result; negative control: no amplification curve appears, or the Tt value is more than or equal to 25min, which is the content of effective result, and the Tt value of each sample is less than 25min, and the sample is judged to be positive.
The result shows that the detection method of the RDA fluorescence detection kit established in the embodiment can detect rotavirus RNA in the excrement.
TABLE 2 establishment of the detection method of the kit
| Negative control | | Sample | 1 | |
|
|
| Tt value | - | 09:57 | 10:22 | 13:46 | 10:57 |
EXAMPLE 2 detection of reagent sensitivity test by RDA fluorescence method
The positive control was a pUC57-NSP3 plasmid containing the NSP3 gene of Rotavirus (RV), and the negative control was an empty vector pUC57 plasmid.
The specific operation is as follows:
step one, the positive control plasmid is diluted to 10^4c, and then diluted to 10^3c, 10^2c and 10^1c respectively by 10 times of gradient dilution.
And step two, sample processing. Respectively taking 5 mu L of the plasmids with each concentration in the step one in an EP tube, simultaneously taking 5 mu L of the negative control in another EP tube, respectively adding 20 mu L of Buffer A, uniformly mixing by shaking, and standing for 10-15min at room temperature;
and step three, preparing and detecting the system. Adding 25 mu L of Buffer B into each tube, vibrating and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence method reaction module, covering a tube cover, vibrating and centrifuging, and immediately detecting; the reaction procedure is as follows: collecting fluorescence signals every minute at 39 ℃ for 1 minute and 30 cycles;
and step four, judging a result. And (3) judging standard:
(ii) positive control: typical amplification curves appear, and the Tt value is less than 25min, which is a valid result;
negative control: no amplification curve appears, or the Tt value is more than or equal to 25min, which is an effective result;
thirdly, the detected sample:
a. if the Tt value is less than 25min, judging the test result to be positive;
b. if the Tt value is more than or equal to 30min, judging the result to be negative;
c. if Tt value is less than or equal to 25min and less than 30min, the result is judged to be suspicious and needs to be repeatedly detected for confirmation; the result of the secondary detection is that the Tt value is more than or equal to 25min and less than 30min, the negative control Tt value is referred to, and if the negative control Tt value is more than or equal to 30min, the positive result is judged.
The results are shown in table 3 and fig. 6. The negative control Tt value is NA, and meets the content of 'no amplification curve appears or Tt value is more than or equal to 25 min' in the determination standard. Tt values of 10^4c, 10^3c, 10^2c and 10^1c are all less than 25min, and according to the result judgment standard, results of 10^4c, 10^3c, 10^2c and 10^1c are positive.
Namely, the sensitivity of the detection kit by the RDA fluorescence method reaches 10 copies.
TABLE 3 sensitivity test results
| |
10^4 | 10^3 | 10^2 | 10^1 | |
| Tt value | - | 09:39 | 12:11 | 12:15 | 14:54 |
Example 3 detection of reagent specificity by RDA fluorescence assay
The specificity of the kit is tested by detecting 4 samples which are clinically collected and verified to be positive corresponding to the virus by fluorescent quantitative PCR (total of 4) of 2 Rotavirus (RV), 1 Norovirus (NV) and 1 Adenovirus (AV).
The specific operation is as follows:
step one, sample processing. Respectively taking 5 mu L of the 4 positive samples in an EP tube, simultaneously taking 5 mu L of the positive control and the negative control of the kit in a new EP tube, respectively adding 20 mu L of Buffer A, shaking and uniformly mixing, and standing at room temperature for 10-15 min;
and step three, preparing and detecting the system. Adding 25 mu L of Buffer B into each tube, vibrating and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence method reaction module, covering a tube cover, vibrating and centrifuging, and immediately detecting; the reaction procedure is as follows: collecting fluorescence signals every minute at 39 ℃ for 1 minute and 30 cycles;
and step four, judging a result. And (3) judging standard:
(ii) positive control: typical amplification curves appear, and the Tt value is less than 25min, which is a valid result;
negative control: no amplification curve appears, or the Tt value is more than or equal to 25min, which is an effective result;
thirdly, the detected sample:
a. if the Tt value is less than 25min, judging the test result to be positive;
b. if the Tt value is more than or equal to 30min, judging the result to be negative;
c. if Tt value is less than or equal to 25min and less than 30min, the result is judged to be suspicious and needs to be repeatedly detected for confirmation; the result of the secondary detection is that the Tt value is more than or equal to 25min and less than 30min, the negative control Tt value is referred to, and if the negative control Tt value is more than or equal to 30min, the positive result is judged.
The results are shown in Table 4. The positive control and the negative control accord with' a positive control: typical amplification curves appear, and the Tt value is less than 25min, which is a valid result; negative control: no amplification curve appeared, or the Tt value was not less than 25mn, which is the content of effective result ". Tt values of the RV samples are all smaller than 25min, and the RV samples are judged to be positive; NV and AV had no amplification curve and were judged negative.
That is, RDA fluorescence detection is positive only when the target pathogen is rotavirus, and negative for other viruses.
TABLE 4 results of specificity test
| Negative control | Positive control | RV-1 | RV-2 | NV | AV | |
| Tt value | - | 08:36 | 08:57 | 10:24 | - | - |
Example 4RDA fluorescence assay kit stability test
The liquid reagent needs to be stored at low temperature and can not be repeatedly frozen and thawed. The RDA fluorescence method reaction module is dried in vacuum to form the powdery reagent in the kit, and the freeze-dried powdery reagent can be stored at normal temperature, so that the cost of cold chain transportation and low-temperature storage is saved, and the operation is simpler. This example demonstrates the stability of the RDA fluorescence detection kit.
The specific operation is as follows:
the eight tubes containing the 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 days, 90 days, and 180 days, respectively.
Step one, sample processing. Respectively taking 5 mu L of positive control/negative control of the kit, respectively adding 20 mu L of Buffer A into an EP tube, shaking and uniformly mixing, and standing at room temperature for 10-15 min;
step two, system preparation and detection. Adding 25 mu L of Buffer B into each tube, vibrating and uniformly mixing, adding 50 mu L of mixed solution into an RDA fluorescence method reaction module, covering a tube cover, vibrating and centrifuging, and immediately detecting; the reaction procedure is as follows: collecting fluorescence signals every minute at 39 ℃ for 1 minute and 30 cycles;
and step three, judging a result. And (3) judging standard:
(ii) positive control: typical amplification curves appear, and the Tt value is less than 25min, which is a valid result;
negative control: no amplification curve appears, or the Tt value is more than or equal to 25min, which is an effective result;
thirdly, the detected sample:
a. if the Tt value is less than 25min, judging the test result to be positive;
b. if the Tt value is more than or equal to 30min, judging the result to be negative;
c. if Tt value is less than or equal to 25min and less than 30min, the result is judged to be suspicious and needs to be repeatedly detected for confirmation; the result of the secondary detection is that the Tt value is more than or equal to 25min and less than 30min, the negative control Tt value is referred to, and if the negative control Tt value is more than or equal to 30min, the positive result is judged.
The results are shown in table 5 and fig. 7, 8, 9 and 10. The reagent freeze-dried powder of the RDA fluorescence method reaction module stored for 0 day, 30 days, 90 days and 180 days is tested respectively, each Tt value is less than 25min, and according to the result judgment standard, the detection results of the reagent in the reagent kit of the patent are positive in 0 day, 30 days, 90 days and 180 days after the reagent is freeze-dried. The reagent in the kit disclosed by the patent can be stably stored for at least 3 months at 37 ℃ after being lyophilized.
TABLE 537 ℃ storage stability
| |
30 days | 90 days | 180 days | |
| Negative control | - | - | - | - |
| Positive control | 09:46 | 10:23 | 10:57 | 11:34 |
Sequence listing
<110> Guangzhou Puchunjun biological science and technology Co., Ltd
<120> RDA method and kit for rapidly detecting rotavirus
<160> 9
<170> SIPOSequenceListing 1.0
<210> 2
<211> 30
<212> DNA
<213> FAM-labeled fluorescent Probe (SEQ ID NO.1)
<400> 2
caacctcagc tcttgagaat atgggaatag 30
<210> 2
<211> 49
<212> DNA
<213> FAM-labeled fluorescent Probe (SEQ ID NO. 2)
<400> 2
gaagctgcag ttgtagctgc aacctcagct cttgagaata tgggaatag 49
<210> 3
<211> 26
<212> DNA
<213> primer sequence (SEQ ID NO. 3)
<400> 3
tggagtctac gcaacagatg gccgtc 26
<210> 4
<211> 27
<212> DNA
<213> primer sequence (SEQ ID NO. 4)
<400> 4
actctagaat atatatcctg ataatca 27
<210> 5
<211> 584
<212> DNA
<213> target sequence (SEQ ID NO. 5)
<400> 5
atgctcaaga tggagtctac gcaacagatg gccgtctcaa ttattaactc ttcttttgaa 60
gctgcagttg tagctgcaac ctcagctctt gagaatatgg gaatagaata tgattatcag 120
gatatatatt ctagagtaaa gaataaattt gattttgtga tggacgattc tggtgttaaa 180
aataatctga ttggtaaagc aataactatt gatcaagctt tgaataataa atttggatct 240
gctataagaa atagaaactg gcttgctgat acttctagag cagctaaatt ggatgaggat 300
gtaaacaaac taagaatgat gttatcatca aaaggaattg atcaaaaaat gagagtttta 360
aacgcatgct tcagtgtaaa aagaatacct ggaaaatcat catctattat taaatgcaca 420
aaattgatgc gtgataaatt ggaacgtggt gaagttgaag tggatgattc atttgtggat 480
gaaaaaatgg aagtggatac cattgactgg aaatcgcgct atgagcaatt ggagcaaagg 540
tttgaatcat tgaaatccag ggtaaatgaa aaatataata attg 584
<210> 6
<211> 1158
<212> DNA
<213> recombinase KX nucleotide sequence (SEQ ID NO. 6)
<400> 6
atgtcaaaca aagcactact aaaaaaactg atcaaaaact cgaatagcca aactgcatct 60
gtactttctg aaagcgacgt attcaacaat attaccatca cgcgaacccg tgtgccgatt 120
ctgaatctgg cgttgtccgg tgcgtttaac ggtggcctaa cttctggtct tacccttttc 180
gctggcccgt ccaaacactt caaatccaac ttaggtttgc ttactgtagc ggcgtatctc 240
aaaacgtatg aagatgctgt gtgcctgttc tacgattcag aaaaaggtgt tactaaatcc 300
tatctgaaat caatgggtgt tgatccggat cgtgttgtgt atactcgtat cacgacggtc 360
gagcagttgc gtaatgacgt tgtaagccag cttaacgcgc ttgaacgcgg tgataaggtg 420
attgtattcg ttgactcagt aggcaacacg gcaagtaaaa aagaacttgc tgacgcgctt 480
tctgataacg ataaacagga tatgacgcga gcaaaagcat taaaaggtat gttccgtatg 540
gttacgcctt atctggctga cctggatatc ccgatggttt gtatctgtca tacctatgac 600
acacaagaaa tgtacagcaa gaaagttatt tctggtggta ctggtttaat gtattccgct 660
gatactgcga tcatcctggg taaacaacag gtgaaagaag gtactgaggt ggtaggttat 720
gatttcatca tgaatatcga aaaatctcga ttcgtgaaag agaaatcaaa attcccgctg 780
catgttacct atgaaggcgg tattagtatg tattctggcc ttttggatct ggcaatggaa 840
atgaactttg tacagaccgt aaccaaaggc tggcgcaacc gcgctttcct gaataccgag 900
actggcgaac tcgaagttga agaaaagaaa tggcgtgagt cagaaacaaa tagcgttgaa 960
ttctggcgtc ctctgtttac tcatcaacca ttcttgaaag ctatcgaaga aaagtataag 1020
atcccagatc gtgaaatcag tgatggttcc gcgctggaag atttatacag cactgatagc 1080
atcccagatc ctgatctgga tgatgacgat atcccagaat catttgatga tatcgaagaa 1140
aacgacgaaa ttttataa 1158
<210> 7
<211> 385
<212> PRT
<213> recombinase KX amino acid sequence (SEQ ID NO. 7)
<400> 7
Met Ser Asn Lys Ala Leu Leu Lys Lys Leu Ile Lys Asn Ser Asn Ser
1 5 10 15
Gln Thr Ala Ser Val Leu Ser Glu Ser Asp Val Phe Asn Asn Ile Thr
20 25 30
Ile Thr Arg Thr Arg Val Pro Ile Leu Asn Leu Ala Leu Ser Gly Ala
35 40 45
Phe Asn Gly Gly Leu Thr Ser Gly Leu Thr Leu Phe Ala Gly Pro Ser
50 55 60
Lys His Phe Lys Ser Asn Leu Gly Leu Leu Thr Val Ala Ala Tyr Leu
65 70 75 80
Lys Thr Tyr Glu Asp Ala Val Cys Leu Phe Tyr Asp Ser Glu Lys Gly
85 90 95
Val Thr Lys Ser Tyr Leu Lys Ser Met Gly Val Asp Pro Asp Arg Val
100 105 110
Val Tyr Thr Arg Ile Thr Thr Val Glu Gln Leu Arg Asn Asp Val Val
115 120 125
Ser Gln Leu Asn Ala Leu Glu Arg Gly Asp Lys Val Ile Val Phe Val
130 135 140
Asp Ser Val Gly Asn Thr Ala Ser Lys Lys Glu Leu Ala Asp Ala Leu
145 150 155 160
Ser Asp Asn Asp Lys Gln Asp Met Thr Arg Ala Lys Ala Leu Lys Gly
165 170 175
Met Phe Arg Met Val Thr Pro Tyr Leu Ala Asp Leu Asp Ile Pro Met
180 185 190
Val Cys Ile Cys His Thr Tyr Asp Thr Gln Glu Met Tyr Ser Lys Lys
195 200 205
Val Ile Ser Gly Gly Thr Gly Leu Met Tyr Ser Ala Asp Thr Ala Ile
210 215 220
Ile Leu Gly Lys Gln Gln Val Lys Glu Gly Thr Glu Val Val Gly Tyr
225 230 235 240
Asp Phe Ile Met Asn Ile Glu Lys Ser Arg Phe Val Lys Glu Lys Ser
245 250 255
Lys Phe Pro Leu His Val Thr Tyr Glu Gly Gly Ile Ser Met Tyr Ser
260 265 270
Gly Leu Leu Asp Leu Ala Met Glu Met Asn Phe Val Gln Thr Val Thr
275 280 285
Lys Gly Trp Arg Asn Arg Ala Phe Leu Asn Thr Glu Thr Gly Glu Leu
290 295 300
Glu Val Glu Glu Lys Lys Trp Arg Glu Ser Glu Thr Asn Ser Val Glu
305 310 315 320
Phe Trp Arg Pro Leu Phe Thr His Gln Pro Phe Leu Lys Ala Ile Glu
325 330 335
Glu Lys Tyr Lys Ile Pro Asp Arg Glu Ile Ser Asp Gly Ser Ala Leu
340 345 350
Glu Asp Leu Tyr Ser Thr Asp Ser Ile Pro Asp Pro Asp Leu Asp Asp
355 360 365
Asp Asp Ile Pro Glu Ser Phe Asp Asp Ile Glu Glu Asn Asp Glu Ile
370 375 380
Leu
385
<210> 8
<211> 420
<212> DNA
<213> KY protein nucleotide sequence (SEQ ID NO. 8)
<400> 8
atgagtttga aattagaaga tctacaaaat gaacttgaaa aggatatgct gatagatccc 60
ctcaagttgc aatcagaatc agcggatatc ccgaagattt gggctaaatg gcttcgatac 120
cattcaaacg ctaagaaaaa attgatccaa cttcatgcga aaaaagaagc tgatgtgaag 180
gatcgtatgt tgtactacac cggaaggcat gacaaagaaa tgtgcgaagt ggtgtatact 240
gggactactg aaattaaaat cgcgatcgct ggggatccga aaattgtaga aaccaacaag 300
ctgatccagt attatgacat ggtggtagat ttcaccagca aagcactgga tatcgtcaaa 360
aacaaaggat actctatcaa aaacatgtta gagatccgta aattagaaag tggtgcataa 420
<210> 9
<211> 139
<212> PRT
<213> KY protein amino acid sequence (SEQ ID NO. 9)
<400> 9
Met Ser Leu Lys Leu Glu Asp Leu Gln Asn Glu Leu Glu Lys Asp Met
1 5 10 15
Leu Ile Asp Pro Leu Lys Leu Gln Ser Glu Ser Ala Asp Ile Pro Lys
20 25 30
Ile Trp Ala Lys Trp Leu Arg Tyr His Ser Asn Ala Lys Lys Lys Leu
35 40 45
Ile Gln Leu His Ala Lys Lys Glu Ala Asp Val Lys Asp Arg Met Leu
50 55 60
Tyr Tyr Thr Gly Arg His Asp Lys Glu Met Cys Glu Val Val Tyr Thr
65 70 75 80
Gly Thr Thr Glu Ile Lys Ile Ala Ile Ala Gly Asp Pro Lys Ile Val
85 90 95
Glu Thr Asn Lys Leu Ile Gln Tyr Tyr Asp Met Val Val Asp Phe Thr
100 105 110
Ser Lys Ala Leu Asp Ile Val Lys Asn Lys Gly Tyr Ser Ile Lys Asn
115 120 125
Met Leu Glu Ile Arg Lys Leu Glu Ser Gly Ala
130 135
Claims (10)
1. A probe for detecting rotavirus is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. The probe for detecting rotavirus of claim 1, wherein the nucleotide sequence is the probe of SEQ ID NO.1, the 5 'end of the probe is marked with a luminescent group, the 3' end of the probe is marked with a quenching group, and any position of the 5 th to 10 th bases is replaced by a tetrahydrofuran residue (THF).
3. The probe for detecting rotavirus of claim 1, wherein the nucleotide sequence is a probe of SEQ ID NO.2, the 30 th base T from the 5 'end is marked with a luminescent group, the 32 th base is replaced by a tetrahydrofuran residue (THF), the 33 th base is marked with a quencher group, and the 3' end is subjected to C3-spacer blocking modification.
4. A group of target sequences, primer pairs and probes for detecting rotavirus are characterized in that the probes are the probes of any one of claims 1 to 3, the nucleotide sequences of the primer pairs are shown as SEQ ID NO.3 and SEQ ID NO.4, and the target sequences are shown as SEQ ID NO. 5.
5. A kit for detecting rotavirus, which is characterized in that the kit comprises a nucleic acid extraction reagent, a constant temperature amplification reaction module, a positive control and a negative control, a probe as claimed in any one of claims 1 to 3 and a primer pair as claimed in claim 4.
6. The kit of claim 5, wherein the isothermal amplification reaction module is a lyophilized powder reagent of a mixed reagent of RDA or RPA isothermal amplification reaction; the freeze-dried powder reagent of the mixed reagent for the RDA constant-temperature amplification reaction comprises recombinase KX with a nucleotide sequence shown in SEQ ID NO. 6.
7. The kit of claim 6, wherein the lyophilized powder reagent of the mixed reagent for RDA isothermal amplification reaction comprises recombinase KX 60-600ng/μ L, KY protein 16-192 ng/μ L, single-stranded binding protein gp 32100-1000 ng/μ L, strand-displacement DNA polymerase 3-100ng/μ L, exonuclease 30-200U, creatine kinase 0.1-0.8mg/ml, creatine phosphate 25-75mM, reverse transcriptase 200U, Tris buffer 20-100mM, PEG 2.5-10%, potassium acetate or sodium acetate 0-150mM, dATP 1-5mM, dNTPs 150-600nM, DTT 1-12mM, the probe 150nM-600nM, and the primer pair 150-600 nM.
8. The kit of claim 5, wherein the nucleic acid extraction reagent comprises Buffer A, Buffer B; the Buffer A is a sample lysate and contains a Tris-HCL Buffer system, NaOH, SDS, EDTA, guanidinium isothiocyanate, Tween80 and triton; the Buffer B contains a Tris Buffer system, potassium chloride and magnesium chloride; the positive control is a plasmid containing rotavirus target genes, and the negative control is an empty vector pUC57 plasmid.
9. A detection method for detecting rotavirus for non-disease diagnosis purpose, which is characterized by comprising the following steps:
extracting nucleic acid of a sample to be detected, performing real-time fluorescence RDA reaction in the presence of a primer pair of rotavirus, a probe, an RDA freeze-dried powder reagent, Buffer A and Buffer B 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 or SEQ ID NO.2, wherein the reaction temperature is 25-42 ℃, and the reaction time is more than 10 minutes.
10. The detection method according to claim 9, wherein the reaction temperature is 39 ℃ and the reaction time is 30 minutes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101660001A (en) * | 2008-08-27 | 2010-03-03 | 中山大学达安基因股份有限公司 | Reagent kit for detecting rotavirus nucleic acid |
| CN107502681A (en) * | 2017-10-12 | 2017-12-22 | 中华人民共和国南通出入境检验检疫局 | A kind of quick real-time fluorescence RT PCR detection kits of A group rotavirus |
| CN110305987A (en) * | 2019-08-06 | 2019-10-08 | 张鹏 | Detect the kit of norovirus I type, norovirus II type and rotavirus |
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2020
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101660001A (en) * | 2008-08-27 | 2010-03-03 | 中山大学达安基因股份有限公司 | Reagent kit for detecting rotavirus nucleic acid |
| CN107502681A (en) * | 2017-10-12 | 2017-12-22 | 中华人民共和国南通出入境检验检疫局 | A kind of quick real-time fluorescence RT PCR detection kits of A group rotavirus |
| CN110305987A (en) * | 2019-08-06 | 2019-10-08 | 张鹏 | Detect the kit of norovirus I type, norovirus II type and rotavirus |
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