CN108034763A - Detect primer, probe and the kit of hepatitis A and Hepatitis E virus - Google Patents
Detect primer, probe and the kit of hepatitis A and Hepatitis E virus Download PDFInfo
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Abstract
The invention discloses recombinase polymeric enzymatic amplification detection hepatitis A and Hepatitis E primed probe group and kit and detection method, including the primer with nucleotide sequence shown in SEQ ID NO.1 4, and the probe containing nucleotide sequence shown in SEQ ID NO.5 6.The disclosure additionally provides a kind of kit of recombinase polymeric enzymatic amplification detection hepatitis A and Hepatitis E virus, and the kit includes the primed probe group described in the disclosure.The Pass through above-mentioned technical proposal disclosure considerably improves sensitiveness, specificity and the simplicity to hepatitis A and Hepatitis E virus detection.
Description
Technical field
This disclosure relates to biological technical field, and in particular, to one kind detection hepatitis A and Hepatitis E virus are with drawing
Thing probe groups and kit and detection method.
Background technology
Hepatitis be it is worldwide pandemic seriously endanger the health even serious disease of life, it is main at present
It is divided into hepatitis A, hepatitis B, hepatitis C, hepatitis D, Hepatitis E, hepatitis G.It can be incited somebody to action according to route of transmission
It is divided into two major classes, wherein the major transmission path of B-mode, the third type, fourth type, hepatitis G for blood born, iatrogenic infection,
Mother-to-baby transmission, spread through sex intercourse, and wherein blood born and spreads through sex intercourse as most common approach.And A type and the main of Hepatitis E pass through
Fecal oral route is propagated, and is mostly large-scale acute infectious disease.
Hepatitis A, Hepatitis E virus the energy long-term surviving in environment and food, but not reproducible, in contaminated environment and food
Viral level in thing propagates irregularities than relatively low (5-100 infectious particles).Therefore identification A type, Hepatitis E are broken out
Viral source has bigger difficulty.Even if the virus of human body intake low dosage may also cause human diseases, seriously endanger
Human health.
Establish and using round pcr hepatitis A virus and hepatitis type B virus are used for quickly detecting both at home and abroad at present
Method.But Standard PCR detection needs accurate instrument and cumbersome test procedure, and it is time-consuming longer, it is difficult to meet non-
The requirement of Site Detection under laboratory environment.Nucleic acid constant-temperature amplification technology (LAMP) nucleic acid constant-temperature amplification skill compared with normal PCR
Expensive PCR instrument is not required in art, can go out purpose fragment by rapid amplifying in a short time, have the advantages that easy, quick, sensitive.
But LAMP detection RNA samples need to set single reverse transcription step, and 60-90min can just complete to react, and SNP is identified
Degree is inadequate, and primer binding zone domain single-site mutant cannot be identified by LAMP.LAMP uses 65 DEG C of constant-temperature amplifications, if region to be detected
G/C content is too low, excessive or secondary structure is complicated, then expanding effect is poor.And LAMP carries out rolling ring using multigroup primer and answers
System, product amount is huge, easily pollutes experimental situation, produces false positive results.
With the mondial size of population increase and flow gradually it is frequent, hepatitis A, viral hepatitis type E in countries in the world in short-term
In be very popular, great outburst will be more frequent.Therefore, for epidemic situation it is quick break out, spread scope is wide the characteristics of, establish spirit
Sensitivity height, high specificity, the hepatitis A that detection time is short, testing cost is low, simple to operate, equipment is of less demanding and penta
Hepatitis virus detection technique, the transmission controe of quick identification and epidemic situation to case have extremely important meaning.Defended to public
Raw and food security aspect prevention and control have important significance.
The content of the invention
The purpose of the disclosure be solve in the prior art to carry out hepatitis A and Hepatitis E it is quick, sensitive and special
The defects of different detection, there is provided the detection primer probe and kit of a kind of hepatitis A and Hepatitis E virus, and establish one
A type liver of the kind based on recombinase polymeric enzymatic amplification (Recombinase Polymerase Amplification, RPA) technology
Scorching and Hepatitis E virus quick, sensitive, special, easy detection method.
To achieve these goals, the disclosure the first aspect provide recombinase polymeric enzymatic amplification detection hepatitis A and
Hepatitis E virus primed probe group, wherein, including the primer with nucleotide sequence shown in SEQ ID NO.1-4, and,
Probe containing nucleotide sequence shown in SEQ ID NO.5-6；
Wherein, in nucleotide sequence shown in SEQ ID NO.5, the 31st bit base is marked with FAM luminophores from 5 ' ends,
Abasic site, the 34th bit base mark quenching group BHQ1 are connected after 32nd bit base；Nucleotides sequence shown in SEQ ID NO.6
In row, the 31st bit base is marked with VIC luminophores from 5 ' ends, and abasic site, the 34th alkali are connected after the 32nd bit base
Disjunction mark note quenching group BHQ1.
Optionally, the primer with nucleotide sequence shown in SEQ ID NO.7-8 is further included, and, contain SEQ ID
The probe of nucleotide sequence shown in NO.9；Wherein, in nucleotide sequence shown in SEQ ID NO.9, the 19th bit base from 5 ' ends
CY5 luminophores are marked with, abasic site, the 25th bit base mark quenching group BHQ3,3 ' ends are connected after the 22nd bit base
End is marked with phosphate group.
Second aspect of the disclosure provides a kind of recombinase polymeric enzymatic amplification detection hepatitis A and Hepatitis E virus
Detection method, wherein, the detection method includes the following steps：
(1) total serum IgE of sample to be tested is extracted；
(2) using the total serum IgE as template, and using the primed probe group described in disclosure the first aspect, recombinated
Enzymatic polymerization enzymatic amplification reacts；
(3) FAM and VIC sense channel fluorescence signals are collected, obtain testing result；
If a) FAM fluorescence channels have amplification curve, judge to contain hepatitis A virus in sample；
If b) VIC fluorescence channels have amplification curve, judge to contain Hepatitis E virus in sample.
Optionally, the final concentration of every primer is respectively 3.5-4.5 μM, and the final concentration of every probe is each
From for 0.8-1.2 μM.
Optionally, the condition of recombinase polymeric enzymatic amplification reaction is included in constant-temperature amplification 5-20min at 25-43 DEG C.
Optionally,
Amplification reaction system includes Quality Control in the positive, reverse transcriptase, reaction system buffer solution, DNA recombinases, DNA gather
At least one of synthase, single strand binding protein, 3 ' -5 ' exonucleases, dNTP and water.
3rd aspect of the disclosure provides a kind of recombinase polymeric enzymatic amplification detection hepatitis A and Hepatitis E virus
Kit, wherein, the kit include disclosure the first aspect described in primed probe group.
The beneficial effects of the present invention are：
What the disclosure was established detects hepatitis A and the dual inspection of Hepatitis E by recombinase polymerase isothermal amplification technique
Survey method, it is quick, comprehensive, quick can to realize that morphology, immunology, LAMP and the detection of substance real-time fluorescence can not be completed
Feel, is special, automatic result judgement, reaching following detection result：
(1) take short
Reverse transcription recombinase polymeric enzymatic amplification technology (RT-RPA) is not required to RNA being converted into cDNA carries out amplified reaction again,
Only need to mix RPA systems with reverse transcriptase, build RT-RPA reaction systems, so that it may directly using RNA as template, realize and reverse
Record is integrated with detection process.Whole reaction 20min can be completed, and the technology such as RT-PCR, Real-time PCR, LAMP
Only process of reverse-transcription just needs the time of 30min, and whole reaction needs 60-90min just to complete.
(2) instrument platform is required low
RPA reactions can carry out at normal temperatures, it is not necessary to be equipped with special thermal cycling amplification instrument, preferably realize A type liver
Scorching and Hepatitis E scene Emergent detection.
(3) specificity is good
RPA can recognize that the SNP of primer binding zone so that it has non-detection target an extremely strong identification capability, and LAMP and
Common Real-time PCR then None- identified SNP, this causes the specificity that RPA is detected to be substantially better than LAMP and Real-time
PCR.The detection method specificity that the present invention is established is also embodied in the specificity of a whole set of primed probe.All primed probes are all
Compare and analyze by Blast, there is the conservative and specificity of height；It can be good at area by specificity experiments verification at the same time
Dividing includes the nucleic acid of hepatitis type B virus, Hepatitis C Virus, Hepatitis D virus, HGV RNA etc., it was demonstrated that detection side
Method has the specificity of height, can accurately distinguish non-detection target and come.
(4) minimum detectability
The minimum detectability for the detection method that the present invention is established can reach 10 copies/reaction.
(5) cost is relatively low
What the present invention was established detects hepatitis A and Hepatitis E method by recombinase polymerase isothermal amplification technique
Human cost and time cost are reduced in operability.This method can be only a step without complicated high end instrument, reaction condition
39 degree, without complex operations.
(6) false negative result is prevented
The IAC added in reaction system of the present invention, can effectively prompt because the reason such as operation error, response inhabitation thing
Caused by false negative testing result.
Other feature and advantage of the disclosure will be described in detail in subsequent specific embodiment part.
Embodiment
The embodiment of the disclosure is described in detail below.It is it should be appreciated that described herein specific
Embodiment is only used for describing and explaining the disclosure, is not limited to the disclosure.
A kind of embodiment of the disclosure provides recombinase polymeric enzymatic amplification detection hepatitis A and Hepatitis E virus
With primed probe group, wherein, including the primer with nucleotide sequence shown in SEQ ID NO.1-4, and, contain SEQ ID
The probe of nucleotide sequence shown in NO.5-6；
Wherein, in nucleotide sequence shown in SEQ ID NO.5, the 31st bit base is marked with FAM luminophores from 5 ' ends,
Abasic site, the 34th bit base mark quenching group BHQ1 are connected after 32nd bit base；Nucleotides sequence shown in SEQ ID NO.6
In row, the 31st bit base is marked with VIC luminophores from 5 ' ends, and abasic site, the 34th alkali are connected after the 32nd bit base
Disjunction mark note quenching group BHQ1.
The specific detection gene of primed probe group selection or conserved sequence of the disclosure are, it is necessary to ensure primed probe section
It is able to cover hepatitis A and Hepatitis E virus virus comprehensively.And different target base is taken into full account in design process
The primed probe of cause is in a reaction system the problem of coamplification, and therefore, what primed probe to be integrated when designing considers Tm
Situations such as being worth uniformity, G/C content homogenization, while avoiding the occurrence of hairpin structure, primer dimer as far as possible, and due to RPA pairs
Primer length requirement is 30-38nt, and the most long 45nt of probe, such sequence easily produces substantial amounts of primer two under constant temperature
Aggressiveness is so as to influence experiment effect, therefore also the design to primer proposes the requirement of higher, to ensure that later stage different primers are visited
The probability that pin expands at the same time.The final a set of special primer probe sequence (referring to table 1) for obtaining the disclosure and providing.
1 hepatitis A of table and Hepatitis E virus RPA detection primer probe summary sheets
The upstream and downstream primer of hepatitis A virus and the nucleotide sequence of probe are detected respectively such as SEQ ID NO.1, SEQ
Shown in ID NO.2, SEQ ID NO.5.
The upstream and downstream primer of hepatitis type B virus and the nucleotide sequence of probe are detected respectively such as SEQ ID NO.3, SEQ
Shown in ID NO.4, SEQ ID NO.6.
The upstream and downstream primer of the positive internal reference of detection and the nucleotide sequence of probe are respectively by SEQ ID NO.7, SEQ ID
Shown in NO.8, SEQ ID NO.9.Wherein, in nucleotide sequence shown in SEQ ID NO.9, the 19th bit base marks from 5 ' ends
There are CY5 luminophores, abasic site, the 25th bit base mark quenching group BHQ3,3 ' ends mark are connected after the 22nd bit base
Note has phosphate group.
In a kind of embodiment of the disclosure, be related to a kind of recombinase polymeric enzymatic amplification (RPA) detection hepatitis A and
The detection method of Hepatitis E virus, wherein, the detection method includes the following steps：
(1) total serum IgE of sample to be tested is extracted；
(2) using the total serum IgE as template, and using the primed probe group described in the disclosure, recombinase polymerase expansion is carried out
Increase reaction；
(3) FAM and VIC sense channel fluorescence signals are collected, obtain testing result；
If a) FAM fluorescence channels have amplification curve, judge to contain hepatitis A virus in sample；
If b) VIC fluorescence channels have amplification curve, judge to contain Hepatitis E virus in sample.
C) if the fluorescence channel of interior Quality Control sample probe is determined as non-specific amplification without obvious amplification curve.
In a kind of embodiment of the disclosure, the primed probe for carrying out interior Quality Control pattern detection is SEQ ID
The primer of nucleotide sequence shown in NO.7-8, and, the probe containing nucleotide sequence shown in SEQ ID NO.9；Also, SEQ
In nucleotide sequence shown in ID NO.9, the 19th bit base is marked with CY5 luminophores from 5 ' ends, is connected after the 22nd bit base
Abasic site, the 25th bit base mark quenching group BHQ3,3 ' end marks have phosphate group.According to CY5 fluorescence channels
Amplification curve is whether there is, determines whether non-specific amplification.
RPA primed probes concentration and reaction system configuration are as follows：
50 μ L of total system, to containing lyophilized enzyme powder (article No.：TAEXO02KIT in 0.2mL TwistAmpExo reaction tubes)
Add 29.5 μ L of rehydration buffer solution, magnesium acetate solution 2.5 μ L (280mmol/L), each 1.5-3 μ L of primer (10 μM), probe
0.2-1 μ L (10 μM), 1 μ L of reverse transcriptase (200U/ μ L), 5 μ L of template, residue are supplied with water.Every primer finally using dense
Degree is respectively 3.5-4.5 μM, and the final concentration of every probe is respectively 0.8-1.2 μM.
In a kind of embodiment of the disclosure, the condition of RPA reactions is included in constant-temperature amplification 5-20min at 25-43 DEG C.
In a kind of particularly preferred embodiment of the disclosure, in order to improve the sensitivity of reaction and specificity, the condition of RPA reactions
It is included in constant-temperature amplification 20min at 39 DEG C.
The direct purpose of the detection method of the disclosure is not to obtain diagnostic result and health status, and simply to having been detached from
The sample of human body or animal body is detected, and what is obtained is merely possible to the information of intermediate result.
The disclosure additionally provides a kind of kit containing foregoing RPA detection primers probe groups.
Preferably, interior Quality Control sequence template is also contained in the kit.
In a kind of embodiment of the disclosure, the kit further includes 5 × reaction system buffer solution, lyophilized recombinase
Powder, lyophilized polymerization enzyme powder, reverse transcriptase, 10 × primed probe mixture, positive control and ultra-pure water.
Hereinafter, the disclosure is further described by embodiment.
Embodiment 1
The present embodiment is used to illustrate hepatitis A and Hepatitis E virus specific primer probe checking test.
(1) synthesis of primer and probe
Entrust the primed probe group 1-3 shown in Reagent Company's synthesis table 2.
Table 2
2 specificity verification of embodiment：
Following sample is selected as simulation interference sample：Norovirus (come from country CDC, 2001), hepatitis type B virus
(come from country CDC, 2005), Hepatitis C Virus (come from country CDC, 2006), salmonella are (in national culture presevation
The heart, CVCC3949), Shigella (from national Culture Collection Center, CVCC3914), staphylococcus aureus is (from country
Culture Collection Center, CVCC1882), bacillus cereus (from national Culture Collection Center, CVCC1782), monocyte increase
Natural disposition listeria spp (from national Culture Collection Center, CVCC1345), yersinia enterocolitica (come from state's house fungus
Kind of collection, CVCC1365), Enterobacter sakazakii (from national Culture Collection Center, CVCC1768), Escherichia coli (come from
National Culture Collection Center, CVCC2356), comma bacillus (from national Culture Collection Center, CVCC3467), Escherichia coli
O157 (from national Culture Collection Center, CVCC7389), Aeromonas hydrophila (from national Culture Collection Center,
CVCC4002 nucleic acid), is assessed, each sample total nucleic acid concentration is 0.001ng/ μ L, and above-mentioned sample is mixed for specificity
As specific detection template, RPA reactions are carried out with primer combination of probe shown in table 1.
RPA reaction systems are prepared：50 μ L of total system, to the 0.2mL TwistAmpExo reaction tube (goods containing lyophilized enzyme powder
Number：TAEXO02KIT rehydration buffer solution 29.5 μ L, magnesium acetate solution 2.5 μ L (280mmol/L), each 2 μ L of primer are added in)
(10 μM), 0.5 μ L of probe (10 μM), 1 μ L of reverse transcriptase (200U/ μ L), 5 μ L of template, residue are supplied with water；
The reaction condition of RPA reactions：Select FAM, VIC and CY5 as follows as reporter group, RPA response procedures：39℃
10s, 39 DEG C of 10s, 39 DEG C of 10s (collection fluorescence), 40 circulations.
RPA reaction results judge：Blank control, IAC and negative control are set up, otherwise invalid regarding experimental result；If FAM leads to
There is amplification curve in road, then the probe of the primer pair of SEQ ID NO.1-2 and SEQ ID NO.5 have non-specific amplification；If VIC leads to
There is amplification curve in road, then the probe of the primer pair of SEQ ID NO.3-4 and SEQ ID NO.6 have non-specific amplification.FAM、VIC
Passage does not occur non-specific amplification.
3 minimum detectability of embodiment is verified：
Concentration is 104The nucleic acid equal proportion mixing of the hepatitis A virus and Hepatitis E virus of copy/μ L is used as template, gradient
It is diluted to 103Copy/μ L, 102Copy/μ L, 10 copies/μ L, L and 1 copy/μ L.According to above-mentioned reaction system response procedures into
Row verification.System is prepared and RPA reaction conditions are the same as the specificity assessment of embodiment 2.So that in the reaction system, viral concentration point
Wei 5 × 104Copy/system, 5 × 103Copy/system, 5 × 102Copy/system, 50 copies/system, 10 copies/μ L and 5 are copied
Shellfish/system.
RPA reaction results judge：Blank control, IAC and negative control are set up, otherwise invalid regarding experimental result；If FAM leads to
There is amplification curve in road, then the probe of the primer pair of SEQ ID NO.1-2 and SEQ ID NO.5 can detect that the template of the concentration；
If VIC passages have amplification curve, the primer pair of SEQ ID NO.3-4 and the probe of SEQ ID NO.5 can detect that the concentration
Template.
This kit of the results show has reached 10 copies/system (2 copies/μ L) for the minimum detectability of target.
4 sample tolerance of embodiment detects
The RNA that hepatitis A virus and Hepatitis E virus are extracted using ethanol precipitation is used as template, according to above-mentioned reaction system with
Response procedures are expanded.
RPA reaction results judge：Blank control, IAC and negative control are set up, otherwise invalid regarding experimental result；If FAM leads to
There is amplification curve in road, then the probe of the primer pair of SEQ ID NO.1-2 and SEQ ID NO.3 can detect that the template of the concentration；
If VIC passages have amplification curve, the primer pair of SEQ ID NO.4-5 and the probe of SEQ ID NO.6 can detect that the concentration
Template.
The template amplification that the results show kit of the present invention is simply extracted with ethanol can obtain positive findings.
5 coverage of embodiment is analyzed
10 plants of hepatitis A virus and Hepatitis E virus are selected respectively as coverage assessment template, nucleic acid concentration 0.001ng/
μ L, are expanded according to reaction system as above and response procedures.
RPA reaction results judge：Blank control, IAC and negative control are set up, otherwise invalid regarding experimental result；If FAM leads to
There is amplification curve in road, then the probe of the primer pair of SEQ ID NO.1-2 and SEQ ID NO.5 can detect that the template；If VIC leads to
There is amplification curve in road, then the probe of the primer pair of SEQ ID NO.3-4 and SEQ ID NO.6 can detect that the template.
The results show kit of the present invention can cover 20 plants of hepatitis A and Hepatitis E virus and detect.
The storage life experiment of 6 kit of embodiment
Assessment template is used as using 100 copies/hepatitis A virus of μ L and the RNA of Hepatitis E virus.
At the 0th day, it is distributed into 10 parts and freezes in -70 DEG C of refrigerators.The kit that establishment finishes is positioned over -20 DEG C of guarantors
Deposit, take the kit of 0,10,15,30,60,90,120,150,180 and 360 day to carry out storage life experiment respectively.Storage life detects
The results are shown in Table 3.
3 storage life result of the test of table
| Storage life | Hepatitis A virus | Hepatitis E virus |
| 0th day | + | + |
| 10th day | + | + |
| 15th day | + | + |
| 30th day | + | + |
| 60th day | + | + |
| 90th day | + | + |
| 120th day | + | + |
| 150th day | + | + |
| 180th day | + | + |
| 360th day | + | + |
As shown in Table 3, kit is stored in -20 DEG C of refrigerators, is the positive in the detection of different storage lives, test result indicates that
The storage life of the kit is at least 1 year.
Comparative example 1
(1) primer, probe synthesis
Sequent synthesis primer and probe according to table 4, the primer and probe are used for RT-RPA detection hepatitis A virus
And Hepatitis E virus.
Table 4 detects the contrast primed probe summary sheet of hepatitis A virus and hepatitis B
(2) specificity verification
Following sample is selected as simulation interference sample：Norovirus (come from country CDC, 2001), hepatitis type B virus
(come from country CDC, 2005), Hepatitis C Virus (come from country CDC, 2006), salmonella are (in national culture presevation
The heart, CVCC3949), Shigella (from national Culture Collection Center, CVCC3914), staphylococcus aureus is (from country
Culture Collection Center, CVCC1882), bacillus cereus (from national Culture Collection Center, CVCC1782), monocyte increase
Natural disposition listeria spp (from national Culture Collection Center, CVCC1345), yersinia enterocolitica (come from state's house fungus
Kind of collection, CVCC1365), Enterobacter sakazakii (from national Culture Collection Center, CVCC1768), Escherichia coli (come from
National Culture Collection Center, CVCC2356), comma bacillus (from national Culture Collection Center, CVCC3467), Escherichia coli
O157 (from national Culture Collection Center, CVCC7389), Aeromonas hydrophila (from national Culture Collection Center,
CVCC4002 nucleic acid), is assessed, each sample total nucleic acid concentration is 0.001ng/ μ L, and above-mentioned sample is mixed for specificity
As specific detection template, the amplification of RT-RPA is carried out.
Reaction system is prepared and response procedures are the same as embodiment 2.
The results show that above reaction result is feminine gender, the primer and probe specificity in comparative example is good.
(3) minimum detectability is verified
Concentration is 104The nucleic acid equal proportion mixing of the hepatitis A virus and Hepatitis E virus of copy/μ L is used as template, gradient
It is diluted to 103Copy/μ L, 102Copy/μ L, 10 copies/μ L, 2 copies/μ L and 1 copy/μ L.According to the anti-of above-mentioned reaction system
Program is answered to be verified.System is prepared and RPA reaction conditions are the same as embodiment 2.So that in the reaction system, viral concentration difference
For 5 × 104Copy/system, 5 × 103Copy/system, 5 × 102Copy/system, 50 copies/system, 10 copies/μ L and 5 are copied
Shellfish/system.
RT-RPA reaction results judge：Blank control, IAC and negative control are set up, otherwise invalid regarding experimental result；If
FAM passages have amplification curve, then the probe of the primer pair of SEQ ID NO.10-11 and SEQ IDNO.12 can detect that the concentration
Template；If VIC passages have amplification curve, the primer pair of SEQ IDNO.13-14 and the probe of SEQ ID NO.15 can detect
Go out the template of the concentration.
Hepatitis A virus and the minimum of Hepatitis E virus are shown in that detection is limited to 100 copies/system in the results show comparative example.
(4) coverage is verified
10 plants of hepatitis A virus and Hepatitis E virus are selected respectively as coverage assessment template, nucleic acid concentration 0.001ng/
μ L, the amplification of RT-RPAR is carried out according to reaction system as above and response procedures.
RT-RPA reaction results judge：If FAM passages have amplification curve, the primer pair and SEQ of SEQ ID NO.10-11
The probe of ID NO.12 can detect that the template；If VIC passages have an amplification curve, the primer pair of SEQ ID NO.13-14 and
The probe of SEQ ID NO.15 can detect that the template.
1 plant of hepatitis A virus of the results show comparative example scheme missing inspection and 2 plants of Hepatitis E virus.
Comparison by embodiment and comparative example can be seen that the detection method that the present invention is established and can disposably examine
Hepatitis A virus and Hepatitis E virus are surveyed, only 20min can complete to detect, and comparative example method needs 90min；This hair
Bright kit can recognize that the single nucleotide mutation point of primer binding zone, there is extremely strong identification capability to non-detection mesh；Present invention examination
Agent box has the specificity of height, can after non-detection target accurately distinguished come, structure decision is simply reliable；This kit pair
Trace detection of nucleic acids ability in sample is stronger.
The preferred embodiment of the disclosure described in detail above, still, the disclosure is not limited in the above embodiment
Detail, in the range of the technology design of the disclosure, a variety of simple variants can be carried out to the technical solution of the disclosure, this
A little simple variants belong to the protection domain of the disclosure.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the disclosure to it is various can
The combination of energy no longer separately illustrates.
In addition, it can also be combined between a variety of embodiments of the disclosure, as long as it is without prejudice to originally
Disclosed thought, it should equally be considered as disclosure disclosure of that.
Sequence table
<110>Beijing Zhuo Cheng Hui Sheng biotech inc
<120>Detect primer, probe and the kit of hepatitis A and Hepatitis E virus
<130> 7071ABT
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 1
ttccagggct ctccccttgc cctaggctct ggcc 34
<210> 2
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 2
agagattcat gaaagccaag ttaacactgc aag 33
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 3
cggttccggc ggtggtttct ggggtgaccg 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 4
ggcctggtca cgccaagcgg agccgagtgg 30
<210> 5
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 5
tgcgcccggc ggggtcaact ccatgattag tnntcatgga gctgtaggag 50
<210> 6
<211> 51
<212> DNA
<213> Artificial Sequence
<400> 6
tgattctcag cccttcgcaa tcccctatat tnnttcatcc aaccaacccc t 51
<210> 7
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 7
tgcggttgct ggcgcctata tcgccgacat c 31
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 8
cagcccagta gtaggttgag gccg 24
<210> 9
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 9
cttcgggctc atgagcgctt tgtttcggcg tggg 34
<210> 10
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 10
gtcaaggctc tcctccatga ttaaggctct g 31
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 11
agagattgcc tacccttgtg gaaag 25
<210> 12
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 12
ctgtaggagt ctaaattggt nntcaacctt gtgtct 36
<210> 13
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 13
accactcggc tgcggtggtc tccgctt 27
<210> 14
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 14
catgggccgg acgccaagcc ggagcga 27
<210> 15
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 15
cacgcgcccc gaatcgctnn tccgctgcct ca 32
Claims (7)
1. recombinase polymeric enzymatic amplification detects hepatitis A and Hepatitis E virus primed probe group, it is characterised in that including
Primer with nucleotide sequence shown in SEQ ID NO.1-4, and, contain nucleotide sequence shown in SEQ ID NO.5-6
Probe；
Wherein, in nucleotide sequence shown in SEQ ID NO.5, the 31st bit base is marked with FAM luminophores from 5 ' ends, and the 32nd
Abasic site, the 34th bit base mark quenching group BHQ1 are connected after bit base；Nucleotide sequence shown in SEQ ID NO.6
In, the 31st bit base is marked with VIC luminophores from 5 ' ends, and abasic site, the 34th bit base are connected after the 32nd bit base
Mark quenching group BHQ1.
2. primed probe group according to claim 1, wherein, further include with nucleotides sequence shown in SEQ ID NO.7-8
The primer of row, and, the probe containing nucleotide sequence shown in SEQ ID NO.9；Wherein, nucleotide shown in SEQ ID NO.9
In sequence, the 19th bit base is marked with CY5 luminophores from 5 ' ends, connects abasic site after the 22nd bit base, the 25th
Kilobase marker quenching group BHQ3,3 ' end marks have phosphate group.
A kind of 3. detection method of recombinase polymeric enzymatic amplification detection hepatitis A and Hepatitis E virus, it is characterised in that institute
Detection method is stated to include the following steps：
(1) total serum IgE of sample to be tested is extracted；
(2) using the total serum IgE as template, and the primed probe group described in usage right requirement 1 or 2, carry out recombinase polymerase
Amplified reaction；
(3) FAM and VIC sense channel fluorescence signals are collected, obtain testing result；
If a) FAM fluorescence channels have amplification curve, judge to contain hepatitis A virus in sample；
If b) VIC fluorescence channels have amplification curve, judge to contain Hepatitis E virus in sample.
4. detection method according to claim 3, wherein, the final concentration of every primer is respectively 3.5-4.5 μM,
The final concentration of every probe is respectively 0.8-1.2 μM.
5. the detection method according to claim 3 or 4, wherein, the condition of recombinase polymeric enzymatic amplification reaction is included in 25-
Constant-temperature amplification 5-20min at 43 DEG C.
6. detection method according to claim 5, wherein, amplification reaction system include Quality Control in the positive, reverse transcriptase,
In reaction system buffer solution, DNA recombinases, archaeal dna polymerase, single strand binding protein, 3 ' -5 ' exonucleases, dNTP and water
It is at least one.
7. recombinase polymeric enzymatic amplification detects the kit of hepatitis A and Hepatitis E virus, it is characterised in that the reagent
Box includes the primed probe group described in claim 1 or 2.
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Cited By (4)
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| CN108841926A (en) * | 2018-07-13 | 2018-11-20 | 锦州医科大学 | A kind of primer, probe and the kit of RT-RPA- Sidestream chromatography double check Hepatitis E virus and hepatitis A virus |
| CN112553374A (en) * | 2020-12-22 | 2021-03-26 | 李轩 | RPA primer for detecting hepatitis E virus based on RPA-LFD method and application thereof |
| CN114540494A (en) * | 2022-02-16 | 2022-05-27 | 中国人民解放军陆军军医大学第二附属医院 | Kit for detecting liver cancer circRNA marker |
| CN115725799A (en) * | 2022-11-15 | 2023-03-03 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting digestive tract pathogens and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841926A (en) * | 2018-07-13 | 2018-11-20 | 锦州医科大学 | A kind of primer, probe and the kit of RT-RPA- Sidestream chromatography double check Hepatitis E virus and hepatitis A virus |
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| CN112553374A (en) * | 2020-12-22 | 2021-03-26 | 李轩 | RPA primer for detecting hepatitis E virus based on RPA-LFD method and application thereof |
| CN114540494A (en) * | 2022-02-16 | 2022-05-27 | 中国人民解放军陆军军医大学第二附属医院 | Kit for detecting liver cancer circRNA marker |
| CN115725799A (en) * | 2022-11-15 | 2023-03-03 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting digestive tract pathogens and application thereof |
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