CN109504800A - A kind of RPA method for detecting universal adenovirus hominis, its primer special and probe and purposes - Google Patents
A kind of RPA method for detecting universal adenovirus hominis, its primer special and probe and purposes Download PDFInfo
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Abstract
The present invention provides RPA detection method, its primer special, probe and its purposes in the detection of universal adenovirus hominis for detecting universal adenovirus hominis.The detection method, its primer special and probe are the conserved sequence designs common based on 3 types, 7 types, 11 types, 21 types, 55 type adenovirus hominis hexon genes, have oligonucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.Novel constant-temperature amplification technique is applied in the detection of universal adenovirus hominis by the present invention for the first time, the enzyme reaction process of DNA replication dna in this method analogue body, it relies on specific enzyme and protein combination (recombinase, single strand binding protein and archaeal dna polymerase) expands DNA profiling, the amplification of specific nucleic acid sequence can be realized under 37 DEG C of constant temperature, amplified production can realize that visualization differentiates by lateral chromatography test strips.The detection method high sensitivity that the present invention establishes, detection limit is up to 101Copy/reaction；High specificity, with other pathogens no cross reaction；It is fast to detect speed, it is easy to operate.
Description
Technical field
A kind of RPA method for detecting universal adenovirus hominis of the present invention, its primer special and probe and purposes belong to
Field of biotechnology is related to the molecular biology of universal adenovirus hominis (human adenovirus, HAdV), is related to a kind of inspection
The method and application thereof for surveying universal adenovirus hominis, in particular to it is a kind of to utilize recombinase polymerase constant-temperature amplification
(Recombinase Polymerase Amplification, RPA) technology quickly detect universal adenovirus hominis method, its
Primer special and probe and application thereof.
Background technique
Adenovirus has 57 kinds of serotypes to human body cause illness at present, can be divided into 7 subgroups of A-G altogether, the virus is in healthy population
In usually easily cause slight self limiting respiratory disease, and can be made a definite diagnosis in clinic in most cases, but adenovirus sometimes
It can cause acute human respiratory tract infection, especially severe pneumonia, teenager and children are high-incidence.Adenovirus hominis can cause regionality
Popular or outbreak of epidemic, but since the clinical manifestation of its infection is usually without specificity, it there is no specificity therapeutic method at present, once
Development is severe pneumonia, then lethality is higher, and especially 3 types, 7 types, 11 types, 21 types, 55 types, infectiousness is strong, poly- in crowd
Collection ground, in school, army, once occur easily to cause group infection and eruption and prevalence.Currently used molecular biology inspection
Survey method has regular-PCR method, quantitative real-time PCR, ring mediated isothermal amplification method etc., however these technologies or dependence are all kinds of
Expensive instrument, or professional is needed to operate, it both can not be universal in common basic health department, more unsuitable field and scene
Live quick diagnosis under the adverse circumstances such as detection.Therefore, one kind is established to be suitable for base's quarantine unit, meet and quickly examine by bed
Disconnected, easy easily development, the accurate detection technique of testing result are of great significance for morning discovery, the early control of epidemic situation.
In recent years, constant temperature nucleic acid amplification technology is developed rapidly, RPA technology be known as be alternative PCR nucleic acid
Detection technique, the technology amplification principle polymerase-mediated based on recombinase, the enzyme reaction process of DNA replication dna in analogue body, according to
Specific enzyme and protein combination (recombinase, single strand binding protein and archaeal dna polymerase) is relied to expand DNA profiling, it can be 25
The amplification of specific nucleic acid sequence is realized under -43 DEG C of constant temperature, and amplified production can be realized by lateral chromatography test strips and be visualized
Differentiate.Requirement of the technology to hardware device is very low, and the reaction time is short, without carrying out complex process to sample, is particularly suitable for
For fields such as in-vitro diagnosis, food safety, bio-safeties.
Summary of the invention
RPA method, its primer special and the probe that it is an object of the present invention to provide a kind of for detecting universal adenovirus hominis
And purposes, it is a kind of 3 types of detection, 7 types, 11 types, 21 types, the universal RPA detection method of 55 type adenovirus hominis, it is to be solved
Technical problem is to provide one group for quickly detecting the specific primer and probe of universal adenovirus hominis, and it is a kind of quickly,
The detection method of universal adenovirus hominis easy, specificity is good.
Therefore present invention detection 3 types, 7 types, 11 types, 21 types, the universal RPA detection method of 55 type adenovirus hominis, first
A purpose is to provide the universal primer for detecting 3 types, 7 types, 11 types, 21 types, 55 type adenovirus hominis, including forward primer and
Reverse primer, totally two.The primer is according to 3 types, 7 types, 11 types, 21 types, 55 type adenovirus hominis hexons (hexon)
Gene design, while DNA homolog sequence is compared and analyzed by software, it further determined 3 types, 7 types, 11 types, 21
The common conserved region of type, 55 type adenovirus hominis hexon genes, nucleotide fragments of this region altogether containing 300 bases, respectively
It is located at the 18418th to 18717 nucleotide site, HAdV7 genome for HAdV3 genome (GenBank:DQ086466.1)
(GenBank:AC_000018.1) it is located at the 18666th to 18965 nucleotide site, there is nucleotide shown in SEQ ID NO.1
Sequence, HAdV11 genome (GenBank:KF528688.1) are located at the 18255th to 18554 nucleotide site, HAdV21 gene
Group (GenBank:KF528688.1) be located at the 18465th to 18764 nucleotide site, HAdV55 genome (GenBank:
FJ643676.1) it is located at a Duan Xulie of the 18233rd to 18532 nucleotide site, there is nucleotide shown in SEQ ID NO.2
Sequence.The screening design specific primer out of SEQ ID NO.1, SEQ ID NO.2 sequence.The specific primer of screening includes just
To primer HAdV-UniF and reverse primer HAdV-UniR11/21/55, it is located at HAdV3 genome the 18458th to 18487
Nucleotide site and the 18679th to 18708 nucleotide site, the 18706th to 18735 nucleotide site of HAdV7 genome and
18927 to 18956 nucleotide sites, the 18295th to 18324 nucleotide site of HAdV11 genome and the 18516th to 18545 core
Thuja acid site, the 18505th to 18534 nucleotide site of 21 genome of HAdV and the 18726th to 18755 nucleotide site,
The 18273rd to 18302 nucleotide site of HAdV55 genome and the 18494th to 18523 nucleotide site, are respectively provided with such as SEQ
Oligonucleotide sequence shown in ID NO.3 and SEQ ID NO.4, the length of primer are 30bp or so, and wherein reverse primer 5 ' is held
It marks biotin (Biotin).The double-stranded DNA obtained after the completion of forward primer and reverse primer amplification will be marked with biotin.
SEQ ID NO.1:
ATGGCCACCCCATCGATGATGCCCCAATGGGCATACATGCACATCGCCGGACAGGATGCTTCGGAGTACCTCA
GTCCGGGTCTGGTGCAGTTCGCCCGTGCAACAGACACCTACTTCAGTATGGGGAACAAATTTAGAAACCCCACAGTG
GCGCCCACCCACGATGTGACCACCGACCGTAGCCAGCGCCTGATGCTGCGCTTCGTGCCCGTTGACCGGGAAGACAA
TACCTACTCTTACAAAGTTCGCTACACGCTGGCTGTAGGCGACAACAGAGTGCTTGACATGGCCAGCACATTC
SEQ ID NO.2:
ATGGCCACCCCATCGATGCTGCCCCAATGGGCATACATGCACATCGCCGGACAGGATGCTTCGGAGTACCTGA
GTCCGGGTCTGGTGCAGTTCGCCCGCGCCACAGACACCTACTTCAATCTGGGAAATAAGTTTAGAAATCCCACCGTA
GCGCCGACCCACGATGTGACCACCGACCGTAGCCAGCGGCTCATGTTGCGCTTCGTGCCCGTTGACCGGGAGGACAA
TACATACTCTTACAAAGTGCGGTACACCCTGGCCGTGGGCGACAACAGAGTGCTGGATATGGCCAGCACGTTC
SEQ ID NO.3:5 ' ACATCGCCGGACAGGATGCTTCGGAGTACC 3 '
SEQ ID NO.4:5 ' GGCCATATCCAGCACTCTGTTGTCGCCCAC 3 '.
The present invention is used to detect the RPA method of universal adenovirus hominis, and second purpose is to provide universal for detecting
The probe of adenovirus hominis.The probe is to be designed according to universal adenovirus hominis hexon gene, while passing through software to gene
Homologous sequence compares and analyzes, and further determined that the conserved region of universal adenovirus hominis hexon gene, this region are contained
The nucleotide fragments of 300 bases have nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2.From SEQ ID
Screening design specific probe in NO.1 and SEQ ID NO.2 sequence, design probe be located at HAdV3 genome the 18496th to
18540 nucleotide sites, the 18743rd to 18787 nucleotide site of HAdV7 genome, HAdV11 genome the 18333rd to
18377 nucleotide sites, the 18543rd to 18587 nucleotide site of HAdV21 genome, HAdV55 genome the 18311st to
18355 nucleotide sites have oligonucleotide sequence shown in SEQ ID NO.5, and 5 ' end mark fluorescent element FAM, 3 ' ends add
Add and extend blocking group (such as phosphate group), the 31st bit base is replaced with into a tetrahydrofuran (THF).
SEQ ID NO.5:
5’ GGTCTGGTGCAGTTCGCCCGTGCAACAGAC[THF]CCTACTTCAGTATGG 3’
The length of the probe is 45bp, wherein 5 ' end 30bp, 3 ' end 15bp.
The probe is by fluorescein (Fluoresceincarboxylic acid FAM), 5 ' terminal sequences, tetrahydrofuran (THF), 3 ' terminal sequences and 3 ' ends
Extend blocking group (phosphate group) composition.
The DNA that label after the probe and amplification has anneals, and the nfo enzyme in RPA system will be cut at the position THF
Disconnected probe enables probe to continue to extend at 3 ' ends in the case where polymerizeing enzyme effect, and the final amplification for obtaining FAM and Biotin double labelling produces
Object.
The present invention is used to detect the RPA method of universal adenovirus hominis, and there is provided be used for universal people for third purpose
The RPA detection method that adenovirus quickly detects, the method are expanded using above-mentioned RPA primer and probe, and combine side
Visualization judgement is carried out to chromatography nucleic acid detection test strip.
The RPA method of the present invention for being used to detect universal adenovirus hominis, comprising the following steps:
(1) using sample to be tested genomic DNA as template, RPA reaction is carried out under the label of the primer sets and probe；
(2) result judgement, the RPA product specially obtained with above-mentioned lateral chromatography nucleic acid detection test strip to step (1) are carried out
It is detected, if detection line and nature controlling line develop the color, judging result is positive findings；If detection line does not develop the color, nature controlling line
Colour developing, judging result is negative findings；If nature controlling line does not develop the color, regardless of whether detection line develops the color, judgement result is invalid.
Preferably, method of the present invention, the specific steps are as follows:
(1) amplifing reagent prepare and sample-adding: by 10 μm of 2.1 μ L of ol/L forward primer, 10 μm of 2.1 μ L of ol/L reverse primer,
10 μm of 0.6 μ L of ol/L probe, 1 μ L of sample to be tested, it is formed in advance without 12.2 μ L of DNase and RNase water and 29.5 μ L of buffer
Mixed 47.5 μ L of liquid, is added to the TwistAmp containing freeze-drying enzyme powder®In nfo reaction tube.Then the magnesium acetate solution of 2.5 μ L is added
Onto reaction lid inner wall;
(2) it expands: the magnesium acetate solution in reaction lid is got rid of down, 37 DEG C of 20 min of amplification after mixing well are reacting 4 min
When, reaction tube taking-up is mixed well, puts back to continue to expand in reaction unit later;
(3) result judges: reaction tube is put into above-mentioned disposable nucleic acid detection apparatus, RPA product is detected, detection line
It develops the color with nature controlling line, judging result is positive findings；Detection line does not develop the color, and nature controlling line colour developing, judging result is negative findings；
Nature controlling line does not develop the color, and regardless of whether detection line develops the color, judgement result is invalid.
Reagent in the RPA method for detecting universal adenovirus hominis for the RPA reaction system of 50 μ L,
The concentration of the forward primer is 10 μm of ol/L, and the concentration of reverse primer is 10 μm of ol/L, and the concentration of the probe is
2.5 μm of ol/L, magnesium ion concentration are 280 μm of ol/L, and template sample-adding amount is 1 μ L.2.1 μ L forward primers, 2.1 μ L are anti-
Premixed liquid is formed without DNase and RNase water and 29.5 μ L buffers to primer, 0.6 μ L probe, 1 μ L sample, 12.2 μ L,
It is added in the TwistAmp nfo reaction tube containing freeze-drying enzyme powder, then the magnesium acetate solution of 2.5 μ L is added to the lid of reaction tube
On son, the magnesium acetate solution on reaction tube lid is got rid of down, 37 DEG C of 20 min of amplification after mixing well, after reacting 4 min,
Reaction tube taking-up is mixed well, puts back to and continues to expand in reaction unit.It is detected, is tried using lateral chromatography test strip
Paper slip developing time is controlled in 3-5 min.
The primer special and/or the probe are for detecting universal adenovirus hominis.
The principle that the present invention is used to detect universal adenovirus hominis is using RPA technology, to the spy of universal adenovirus hominis
The conservative target sequence of the opposite sex, i.e., the conserved sequence of universal adenovirus hominis hexon gene are detected, which can be used as universal
One of marker gene of adenovirus hominis.
The present invention is used to detect the RPA method of universal adenovirus hominis, has saved the detection time of universal adenovirus hominis,
Detection can complete amplification at 37 DEG C in 20 min, entire detection process can complete in 1 hour, with Standard PCR and real-time glimmering
Fluorescent Quantitative PCR, which needs a few hours to compare, highly shortened detection time.
The present invention is used to detect the RPA method of universal adenovirus hominis, reduces reaction temperature, RPA only needs constant temperature 37
DEG C experiment can be completed, 63 DEG C of the temperature well below 60-95 DEG C and LAMP of quantitative fluorescent PCR.
The present invention is used to detect the RPA method of universal adenovirus hominis, and method is simpler, easy to carry: needed for amplification
Enzyme and some other requirement preservation can be lyophilized, can place for a long time at normal temperature, when amplification only needs that water is added
Buffer, primer, probe and template are solved, and magnesium ion starting reaction is added, and sample does not need to carry out complex reaction.
The present invention is used to detect the RPA method of universal adenovirus hominis, and sensitivity is high, high specificity, can on site or
Immediately detection, has broad application prospects.
Detailed description of the invention
Below with reference to attached drawing, the invention will be further described:
Fig. 1 shows the testing result of different primers combination in the optimal primer combined sorting of RPA detection architecture.Every group of combination is respectively provided with
One group of negative control, as shown, the plasmid of (1) detection is HAdV3/7 positive plasmid, the plasmid of (2) detection is test result
HAdV11/21/55 positive plasmid, NC represent negative control.
Fig. 2 is to determine the optimal reverse primer and probe combined concentration for detecting universal adenovirus hominis RPA detection method.If
The concentration gradient for determining reverse primer is 10 μm of ol/L, 5 μm of ol/L, 2.5 μm of ol/L, and the concentration of probe is 10 μm of ol/L, 5 μm of ol/
L, 2.5 μm of ol/L, the probe by the reverse primer of three kinds of concentration respectively with three kinds of concentration are combined, and are combined into 9 groups of combination (groups
3) number of compiling in collaboration with is shown in Table, every group of combination is respectively provided with one group of negative control, and test result is as shown, the plasmid of (1) detection is
The plasmid of HAdV3/7 positive plasmid, (2) detection is HAdV11/21/55 positive plasmid, and NC represents negative control.
Fig. 3 is to determine the best proliferation time for detecting universal adenovirus hominis RPA detection method.Proliferation time is set as 5
Min, 10 min, 15 min, 20 min, and it is provided with a negative control, test result is as shown, the plasmid that (1) is detected
Plasmid for HAdV3/7 positive plasmid, (2) detection is HAdV11/21/55 positive plasmid, and NC represents negative control.
Fig. 4 is the sensitivity for detecting universal adenovirus hominis RPA method, carries out ten after quantifying to two kinds of positive plasmids
Times doubling dilution, obtaining concentration is 1010-100 Copy/μ L Plasmid DNA, is tested, experimental condition is as subsequent template
Above-mentioned optimum test condition, test result as shown, the plasmid of (1) detection is HAdV3/7 positive plasmid, detect by (2)
Plasmid is HAdV11/21/55 positive plasmid, and NC represents negative control.
Fig. 5 is the specificity for detecting universal adenovirus hominis RPA method, respectively with HAdV3, HAdV7, HAdV11,
The diseases such as HAdV21 positive plasmid, chlamydia psittaci, staphylococcus aureus, Coxiella burnetii, Streptococcus suis, Escherichia coli
The sample of pathogen DNA and healthy person DNA are that template is tested, and test result is as shown in the figure.
Specific embodiment
It is following that examples are only for illustrating the present invention and not for limiting the scope of the present invention.It is not specified in the following example
The experimental method of actual conditions, usually according to normal condition as Sambrook et al. compiles institute in " Molecular Cloning:A Laboratory guide "
The condition stated, or the condition suggested according to manufacturer.
The acquirement approach of various biomaterials described in embodiment is to provide a kind of approach of experiment acquisition only to reach
To specifically disclosed purpose, the limitation to biological material source of the present invention should not be become.In fact, used biomaterial
Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to mentioning in embodiment
Show and is replaced.
RPA primer and probe used is synthesized by Nanjing Genscript Biotechnology Co., Ltd., and it is equal that all sequences measure work
It is completed by Nanjing Genscript Biotechnology Co., Ltd..
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Embodiment 1: the design and screening of universal adenovirus hominis RPA detection primer and probe
(1) design of primer and probe
Inventor analyzes by literature search and determines that the present invention uses the specific sequence in universal adenovirus hominis hexon gene
It is classified as target gene.Known templet gene sequence, i.e. nucleotides sequence shown in SEQ ID NO.1 are obtained from ncbi database
Column, and above-mentioned sequence is synthesized by Nanjing Genscript Biotechnology Co., Ltd., as positive plasmid, visited in subsequent primer
It is used during needle screening, optimization of reaction system etc. as template.According to RPA primer and probe design principle, 2 groups of design is drawn
Physical prospecting needle, as shown in table 1.
1 primer of table and probe sequence
(2) primer screening
Sequence shown in the artificial synthesized SEQ ID NO.1 and SEQ ID NO.2 containing universal adenovirus hominis hexon gene
Primer and probe are carried out global combinatorial into 4 groups of primer combination of probe, combination number is such as using this plasmid as template by positive plasmid
Shown in table 2.4 groups of primer combination of probe carry out carrying out RPA amplification under the conditions of 37 DEG C respectively, with lateral chromatography detection of nucleic acids test paper
Detection line colour developing situation is index, is filtered out under the conditions of 37 DEG C, the highest primer combination of probe of amplification efficiency, for subsequent
The evaluation and application of RPA detection.
The RPA reaction system of 50 μ L of screening is as follows: 10 μm of 2.1 μ L of ol/L forward primer, 10 μm of ol/L are anti-
To primer 2 .1 μ L, 10 μm of 0.6 μ L of ol/L probe, 1 × 104 Copy/1 μ L of μ L template, without 12.2 μ of DNase and RNase water
L and 29.5 μ L of buffer forms 47.5 μ L of premixed liquid, is added to the TwistAmp containing freeze-drying enzyme powder®In nfo reaction tube, then
The magnesium acetate solution of 2.5 μ L is added on the inner wall of reaction lid.In view of RPA reaction sensitivity is higher, easily there is false sun
Property, inventor is reacted provided with one group of negative control, negative control using empty plasmid carrier.Amplification: by reaction lid
On magnesium acetate solution get rid of down, mix well after 37 DEG C of 20 min of amplification, when react 4 min, by reaction tube taking-up sufficiently
It mixes, puts back to continue to expand in reaction unit later.As a result judge: reaction tube being put into and includes lateral chromatography test strip
It develops the color in disposable nucleic acid detection apparatus, carries out visualization differentiation.A multiple holes are arranged in each reaction.
2 primer combination of probe of table number
The lateral chromatography detection of nucleic acids the result is shown in Figure 1 of 4 groups of primer combination of probe.Fig. 1 is detection time 4 groups of primers when being 20 min
The display situation of probe combinations, the detection line of the lateral chromatography nucleic acid detection test strip of negative control and nature controlling line.
The present invention is used to detect the RPA method of universal adenovirus hominis, and determining primer combination of probe includes: reverse primer
(HAdV-UniR11/21/55) and probe (HAdV-UniP3/7) it, totally two, is respectively provided with such as SEQ ID NO.3 and SEQ ID
Oligonucleotide sequence shown in NO.4.
(3) determination of probe
The probe probe that table 1 is listed be the present invention preferably, the length of probe is 45 bp, wherein 5 ' ends share 30 bp to THF,
3 ' ends share 15 bp to THF.
Probe is extended by fluorescein (Fluoresceincarboxylic acid FAM), 5 ' terminal sequences, tetrahydrofuran (THF), 3 ' terminal sequences and 3 ' ends
A few part compositions of blocking group (phosphate group).Probe has oligonucleotide sequence shown in SEQ ID NO.4, and 5 ' end labels
Fluorescein FAM, 3 ' end additions extend blocking group (such as phosphate group), the 31st bit base are replaced with tetrahydrofuran (THF).Institute
The DNA that label after stating probe and amplification has anneals, and the nfo enzyme in RPA system will cut off probe at the position THF, makes
Probe can continue to extend in the case where polymerizeing enzyme effect at 3 ' ends, the final amplified production for obtaining FAM and Biotin double labelling.
Embodiment 2: universal adenovirus hominis RPA reaction system, amplification and testing conditions optimization
During primer screening, lateral chromatography nucleic acid detection test strip is the case where there are still false positives in detection, therefore
RPA reaction system, amplification and testing conditions need to be optimized
(1) primed probe concentration
The concentration gradient of reverse primer is set as 10 μm of ol/L, 5 μm of ol/L, 2.5 μm of ol/L, the concentration of probe is 10 μ
Mol/L, 5 μm of ol/L, 2.5 μm of ol/L, the probe by the reverse primer of three kinds of concentration respectively with three kinds of concentration are combined, group
9 groups of combinations are synthesized, every group of combination is respectively provided with one group of negative control.Respectively under the conditions of 37 DEG C carry out RPA amplification, amplification finish with
Lateral chromatography nucleic acid detection test strip detection line develops the color situation for index.It is best to filter out expanding effect, and false positive does not occur
Combination.
3 reverse primer concentration of table and concentration and probe concentration combination number
By to lateral chromatography nucleic acid detection test strip Analysis of test results (see figure 2), and consider economic factor, present invention determine that
Reverse primer concentration be 10 μm of ol/L, the concentration of probe is 2.5 μm of ol/L.
(2) optimization of RPA proliferation time
During primer screening, lateral chromatography nucleic acid detection test strip the case where there are still false positives when detecting, therefore
RPA proliferation time need to be optimized.
The RPA reaction system of 50 μ L is as follows: by 10 μm of 2.1 μ L of ol/L forward primer, 10 μm of ol/L reverse primers 2.1
μ L, 10 μm of 0.6 μ L of ol/L probe, 1 × 103 Copy/1 μ L of μ L template, without 12.2 μ L of DNase and RNase water and buffer
29.5 μ L form 47.5 μ L of premixed liquid, are added to the TwistAmp containing freeze-drying enzyme powder®In nfo reaction tube, then by 2.5 μ L
Magnesium acetate solution be added on reaction lid inner wall.Amplification: the magnesium acetate solution in reaction lid is got rid of down, 37 after mixing well
DEG C amplification 5 min, 10 min, 15 min, 20 min, when react 4 min, by reaction tube take out mix well, Zhi Houfang
It returns in reaction unit and continues to expand.Inventor is provided with one group of negative control, and negative control is using empty plasmid carrier as template.Knot
Fruit judgement: reaction tube being put into the disposable nucleic acid detection apparatus for include lateral chromatography test strip and developed the color, and is carried out visual
Change and differentiates.
By to lateral chromatography nucleic acid detection test strip Analysis of test results (see figure 3), two when proliferation time is 20 min
Kind of positive plasmid detection line develop the color clearly, reaches test requirements document, present invention determine that proliferation time be 20 min.
To sum up, it is optimized by RPA amplification and testing conditions, this discovery determination is drawn in the downstream using 10 μm of ol/L
20 min of probe amplification of object and 2.5 μm of ol/L, test strips developing time are controlled in 3-5 min, and detection effect is best.
Embodiment 3: the detection of universal adenovirus hominis RPA detection limits evaluation
By 3/7 type positive plasmid, 11/21/55 type positive plasmid by ten times of doubling dilutions at 1010 - 100One systems such as copy/μ L
Column various concentration, respectively takes 1010、109、108、104、103、102、1011 μ L of copy/μ L positive plasmid is separately added into embodiment 2
Identified reaction system is combined using the primer filtered out, and the reaction condition determined using embodiment 2 is to above-mentioned different copies
Several templates carries out RPA detection, the detection limit of observation RPA detection.
As a result Fig. 4 is seen, from 101Copy/μ L starts both the above positive plasmid and is positive, and negative control is negative, and says
The detection of bright RPA detection method of the present invention is limited to 101Copy/reaction.
Embodiment 4: the Evaluation on specificity of universal adenovirus hominis RPA detection
Evaluation on specificity is with chlamydia psittaci, staphylococcus aureus, Coxiella burnetii, Streptococcus suis, Escherichia coli etc.
Pathogen DNA and healthy person DNA is control, to determine the specificity of RPA detection method of the present invention.
Respectively with HAdV3, HAdV7, HAdV11, HAdV21 positive plasmid, chlamydia psittaci, staphylococcus aureus,
The pathogen such as Coxiella burnetii, Streptococcus suis, Escherichia coli DNA and healthy person DNA is template, using following reaction system:
By 10 μm of 2.1 μ L of ol/L forward primer, 10 μm of 2.1 μ L of ol/L reverse primer, 10 μm of 0.6 μ L of ol/L probe, 1 μ L sample
Originally, 47.5 μ L of premixed liquid is formed without 12.2 μ L of DNase and RNase water and 29.5 μ L of buffer, be added to containing freeze-drying enzyme powder
TwistAmp®In nfo reaction tube,.Then the magnesium acetate solution of 2.5 μ L is added on reaction lid inner wall.Amplification: it will react
Magnesium acetate solution in pipe lid is got rid of down, and 37 DEG C of 20 min of amplification take out reaction tube when reacting 4 min after mixing well
It mixes well.As a result judge: reaction tube is put into the disposable nucleic acid detection apparatus for include lateral chromatography test strip into
Row visualization differentiates that detection line and nature controlling line develop the color, and judging result is positive findings；Detection line does not develop the color, nature controlling line colour developing,
Judging result is negative findings；Nature controlling line does not develop the color, and regardless of whether detection line develops the color, judgement result is invalid.
As a result see Fig. 5, chlamydia psittaci, staphylococcus aureus, Coxiella burnetii, Streptococcus suis, Escherichia coli
Etc. pathogen DNA and healthy person DNA sample detection line do not develop the color, be negative, HAdV3, HAdV7, HAdV11, HAdV21 are positive
The colour developing of plasmid pattern detection line, is positive, and illustrates RPA detection method of the present invention to HAdV3, HAdV7, HAdV11, HAdV21 sun
Property grain, HAdV55 have very strong specificity.
Sequence table
<110>Li Yuexi
<120>a kind of RPA method for detecting universal adenovirus hominis, its primer special and probe and purposes
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 300
<212> DNA
<213>universal adenovirus hominis specific gene sequences (SIPOSequenceListing 1.0)
<220>
<221> misc_feature
<222> (1)..(300)
<400> 1
atggccaccc catcgatgat gccccaatgg gcatacatgc acatcgccgg acaggatgct 60
tcggagtacc tcagtccggg tctggtgcag ttcgcccgtg caacagacac ctacttcagt 120
atggggaaca aatttagaaa ccccacagtg gcgcccaccc acgatgtgac caccgaccgt 180
agccagcgcc tgatgctgcg cttcgtgccc gttgaccggg aagacaatac ctactcttac 240
aaagttcgct acacgctggc tgtaggcgac aacagagtgc ttgacatggc cagcacattc 300
<210> 2
<211> 30
<212> DNA
<213>the forward primer HAdV-UniF in universal adenovirus hominis specific gene sequences
(SIPOSequenceListing 1.0)
<220>
<221> misc_feature
<222> (1)..(30)
<400> 2
acatcgccgg acaggatgct tcggagtacc 30
<210> 3
<211> 30
<212> DNA
<213>the reverse primer HAdV-UniR11/21/55 in universal adenovirus hominis specific gene sequences
(SIPOSequenceListing 1.0)
<220>
<221> misc_feature
<222> (1)..(30)
<223>5 ' end label biotins
<400> 3
ggccatatcc agcactctgt tgtcgcccac 30
<210> 4
<211> 45
<212> DNA
<213>the probe HAdV-UniP3/7 in universal adenovirus hominis specific gene sequences
(SIPOSequenceListing 1.0)
<220>
<221> misc_feature
<222> (1)..(45)
<223>5 ' end mark fluorescent element FAM, 3 ' end additions extend blocking group (such as phosphate group), the 31st bit base are replaced
At a tetrahydrofuran (THF)
<400> 4
ggtctggtgc agttcgcccg tgcaacagac cctacttcag tatgg 45
Claims (10)
1. it is a kind of for detecting the RPA primer special of universal adenovirus hominis, it is according to universal adenovirus hominis hexon gene
Conserved sequence design, the universal adenovirus hominis hexon gene conserved sequence has SEQ ID NO.1 and SEQ ID
Nucleotide sequence shown in NO.2
SEQ ID NO.1:
ATGGCCACCCCATCGATGATGCCCCAATGGGCATACATGCACATCGCCGGACAGGATGCTTCGGAGTACCTCA
GTCCGGGTCTGGTGCAGTTCGCCCGTGCAACAGACACCTACTTCAGTATGGGGAACAAATTTAGAAACCCCACAGTG
GCGCCCACCCACGATGTGACCACCGACCGTAGCCAGCGCCTGATGCTGCGCTTCGTGCCCGTTGACCGGGAAGACAA
TACCTACTCTTACAAAGTTCGCTACACGCTGGCTGTAGGCGACAACAGAGTGCTTGACATGGCCAGCACATTC
SEQ ID NO.2:
ATGGCCACCCCATCGATGCTGCCCCAATGGGCATACATGCACATCGCCGGACAGGATGCTTCGGAGTACCTG
AGTCCGGGTCTGGTGCAGTTCGCCCGCGCCACAGACACCTACTTCAATCTGGGAAATAAGTTTAGAAATCCCACCG
TAGCGCCGACCCACGATGTGACCACCGACCGTAGCCAGCGGCTCATGTTGCGCTTCGTGCCCGTTGACCGGGAGGA
CAATACATACTCTTACAAAGTGCGGTACACCCTGGCCGTGGGCGACAACAGAGTGCTGGATATGGCCAGCACGTTC。
2. according to claim 1 for detecting the RPA primer special of universal adenovirus hominis, it is characterised in that: described to draw
Forward primer in object has oligonucleotide sequence shown in SEQ ID NO.3, and reverse primer has shown in SEQ ID NO.4
Oligonucleotide sequence, and 5 ' end labels biotin (Biotin)；
SEQ ID NO.3:5 ' ACATCGCCGGACAGGATGCTTCGGAGTACC 3 '
SEQ ID NO.4:5 ' GGCCATATCCAGCACTCTGTTGTCGCCCAC 3 '.
3. a kind of RPA probe for detecting according to universal adenovirus hominis, it is characterised in that: be according to claim 1 in it is logical
With type adenovirus hominis hexon gene conserved sequence SEQ ID NO.1 design, which has widow shown in SEQ ID NO.4
Nucleotide sequence, and 5 ' end mark fluorescent elements, 3 ' end additions extend blocking group, the 31st bit base are replaced with tetrahydrofuran
(THF)；
SEQ ID NO.4:
5’GGTCTGGTGCAGTTCGCCCGTGCAACAGAC[THF]CCTACTTCAGTATGG3’。
4. the RPA probe according to claim 3 for detecting according to universal adenovirus hominis, it is characterised in that: the spy
Needle extends blocking group by fluorescein, 5 ' terminal sequences, base analogue tetrahydrofuran (THF), 3 ' terminal sequences and 3 ' ends and forms.
5. the RPA probe according to claim 3 for detecting according to universal adenovirus hominis, it is characterised in that: described
Fluorescein is other fluoresceins of Fluoresceincarboxylic acid FAM or FITC, and the extension blocking group is phosphate group or other blockings
Group.
6. according to claim 3 for detecting RPA probe according to universal adenovirus hominis, it is characterised in that: length is
45 bp, wherein 5 ' 30 bp of end, 3 ' 15 bp of end.
7. a kind of for detecting the RPA method of universal adenovirus hominis, which comprises the following steps:
(1) using sample to be tested genomic DNA as template, in primer special as claimed in claim 2 and/or claim 3 ~ 6 institute
The probe label stated is lower to carry out RPA reaction；
(2) result judgement, the RPA product specially obtained with above-mentioned lateral chromatography nucleic acid detection test strip to step (1) are carried out
It is detected, if detection line and nature controlling line develop the color, judging result is positive findings；If detection line does not develop the color, nature controlling line
Colour developing, judging result is negative findings；If nature controlling line does not develop the color, regardless of whether detection line develops the color, judgement result is invalid.
8. according to claim 7 for detecting the RPA method of universal adenovirus hominis, which is characterized in that including specific
Steps are as follows:
Amplifing reagent prepares and sample-adding: by 10 μm of 2.1 μ L of ol/L forward primer, 10 μm of 2.1 μ L of ol/L reverse primer, 10 μ
0.6 μ L of mol/L probe, 1 μ L of sample to be tested, premixed liquid is formed without 12.2 μ L of DNase and RNase water and 29.5 μ L of buffer
47.5 μ L are added to the TwistAmp containing freeze-drying enzyme powder®In nfo reaction tube;
Then the magnesium acetate solution of 2.5 μ L is added on reaction lid inner wall;
(2) it expands: the magnesium acetate solution in reaction lid is got rid of down, 37 DEG C of 20 min of amplification after mixing well are reacting 4 min
When, reaction tube taking-up is mixed well, puts back to continue to expand in reaction unit later;
(3) result judges: reaction tube is put into above-mentioned disposable nucleic acid detection apparatus, RPA product is detected, detection line
It develops the color with nature controlling line, judging result is positive findings；Detection line does not develop the color, and nature controlling line colour developing, judging result is negative findings；
Nature controlling line does not develop the color, and regardless of whether detection line develops the color, judgement result is invalid.
9. according to claim 7 for detecting the RPA method of universal adenovirus hominis, which is characterized in that the method
In for 50 μ L RPA reaction system reagent, the concentration of the forward primer is 10 μm of ol/L, the concentration of reverse primer
For 10 μm of ol/L, the concentration of the probe is 2.5 μm of ol/L, and magnesium ion concentration is 280 μm of ol/L, and template sample-adding amount is 1
μL;
By 2.1 μ L forward primers, 2.1 μ L reverse primers, 0.6 μ L probe, 1 μ L sample, 12.2 μ L without DNase and RNase
Water and 29.5 μ L buffers form premixed liquid, are added in the TwistAmp nfo reaction tube containing freeze-drying enzyme powder, then by 2.5
The magnesium acetate solution of μ L is added on the lid of reaction tube, and the magnesium acetate solution on reaction tube lid is got rid of down, 37 after mixing well
DEG C amplification 20 min, after react 4 min, by reaction tube take out mix well, put back to and continue to expand in reaction unit, use side
It is detected to chromatography detecting test paper strip, test strips developing time is controlled in 3-5 min.
10. probe described in primer special as claimed in claim 2 and/or claim 3 ~ 6 is for detecting universal people's gland
Virus.
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