CN108034763B - Primer, probe and kit for detecting hepatitis A virus and hepatitis E virus - Google Patents
Primer, probe and kit for detecting hepatitis A virus and hepatitis E virus Download PDFInfo
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Abstract
The invention discloses a primer probe set, a kit and a detection method for detecting hepatitis A and hepatitis E by recombinase polymerase amplification, which comprise a primer with a nucleotide sequence shown by SEQ ID NO.1-4 and a probe containing a nucleotide sequence shown by SEQ ID NO. 5-6. The present disclosure also provides a kit for detecting hepatitis a and hepatitis e viruses by recombinase polymerase amplification, wherein the kit comprises the primer probe set disclosed by the present disclosure. By adopting the technical scheme, the method obviously improves the sensitivity, specificity and simplicity of detection on hepatitis A virus and hepatitis E virus.
Description
Technical Field
The disclosure relates to the field of biotechnology, in particular to a primer probe set for detecting hepatitis A and hepatitis E viruses, a kit and a detection method.
Background
Hepatitis is a serious disease which is widely spread throughout the world and seriously harms human health and even lives, and is mainly classified into hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E and hepatitis G at present. The hepatitis B, C, D and G can be divided into two main categories according to the transmission path, wherein the main transmission paths of the hepatitis B, C, D and G are blood transmission, iatrogenic transmission, maternal and infant transmission and sexual transmission, and the blood transmission and the sexual transmission are the most common paths. Hepatitis A and E are mainly transmitted through a fecal-oral route and are mostly acute infectious diseases in a large range.
Hepatitis A and hepatitis E viruses can survive for a long time in the environment and food, but cannot replicate, and have low virus content (5-100 infected particles) in polluted environment and food and irregular transmission. Therefore, it is difficult to identify the source of hepatitis A and E outbreak virus. Even low doses of the virus ingested by humans can cause human disease and severely harm human health.
At present, the method for rapidly detecting the hepatitis A virus and the hepatitis B virus by adopting the PCR technology is established at home and abroad. However, the conventional PCR detection requires precise instruments and complicated test procedures, and is time-consuming and difficult to meet the requirements of field detection in non-laboratory environments. Compared with the traditional PCR, the nucleic acid isothermal amplification technology (LAMP) does not need an expensive PCR instrument, can rapidly amplify the target fragment in a short time, and has the advantages of simplicity, rapidness, sensitivity and the like. However, the LAMP detection of the RNA sample needs a separate reverse transcription step, the reaction can be completed within 60-90min, the SNP recognition degree is not enough, and the single-point mutation of the primer binding region cannot be recognized by LAMP. LAMP adopts constant temperature amplification at 65 ℃, and if the GC content of the region to be detected is too low or too high or the secondary structure is complex, the amplification effect is poor. And LAMP adopts a plurality of groups of primers for rolling circle replication, so that the product quantity is huge, the experimental environment is easily polluted, and a false positive result is generated.
With the increasing and frequent population worldwide, the prevalence and outbreak of hepatitis A and hepatitis E in the world will be more frequent in a short time. Therefore, aiming at the characteristics of rapid outbreak and wide spread range of epidemic situation, the hepatitis A and hepatitis E virus detection technology which has high sensitivity, strong specificity, short detection time, low detection cost, simple and convenient operation and low equipment requirement is established, and has very important significance for rapid identification of cases and spread control of epidemic situation. Has important value significance for prevention and control in public health and food safety.
Disclosure of Invention
The purpose of the present disclosure is to solve the defect that the prior art can not carry out rapid, sensitive and specific detection on hepatitis A and hepatitis E, provide a primer probe and a kit for detecting hepatitis A and hepatitis E viruses, and establish a rapid, sensitive, specific and simple detection method for hepatitis A and hepatitis E viruses based on Recombinase Polymerase Amplification (RPA) technology.
In order to achieve the above objects, a first aspect of the present disclosure provides a recombinase polymerase amplification primer probe set for detecting hepatitis a and hepatitis e viruses, including a primer having a nucleotide sequence represented by SEQ ID nos. 1 to 4, and a probe having a nucleotide sequence represented by SEQ ID nos. 5 to 6;
wherein, in the nucleotide sequence shown in SEQ ID NO.5, the 31 st base from the 5' end is marked with FAM luminous group, the 32 nd base is connected with abasic site, and the 34 th base is marked with quenching group BHQ 1; in the nucleotide sequence shown in SEQ ID NO.6, the 31 st base from the 5' end is marked with a VIC luminescent group, the 32 nd base is connected with an abasic site, and the 34 th base is marked with a quenching group BHQ 1.
Optionally, the kit also comprises a primer with a nucleotide sequence shown in SEQ ID NO.7-8 and a probe with a nucleotide sequence shown in SEQ ID NO. 9; wherein, in the nucleotide sequence shown in SEQ ID NO.9, the 19 th base from the 5 'end is marked with a CY5 luminescent group, the 22 th base is connected with an abasic site, the 25 th base is marked with a quenching group BHQ3, and the 3' end is marked with a phosphate group.
In a second aspect of the present disclosure, a method for detecting hepatitis a and hepatitis e virus by recombinase polymerase amplification is provided, wherein the method comprises the following steps:
(1) extracting total RNA of a sample to be detected;
(2) performing a recombinase polymerase amplification reaction using the total RNA as a template and the primer probe set of the first aspect of the disclosure;
(3) collecting fluorescence signals of FAM and VIC detection channels to obtain detection results;
a) if the FAM fluorescence channel has an amplification curve, judging that the sample contains hepatitis A virus;
b) if the VIC fluorescence channel has an amplification curve, the sample is judged to contain the hepatitis E virus.
Alternatively, the final use concentration of each primer is 3.5-4.5. mu.M, and the final use concentration of each probe is 0.8-1.2. mu.M.
Optionally, the conditions for the recombinase polymerase amplification reaction include isothermal amplification at 25-43 ℃ for 5-20 min.
Alternatively to this, the first and second parts may,
the amplification reaction system comprises at least one of positive internal quality control, reverse transcriptase, reaction system buffer solution, DNA recombinase, DNA polymerase, single-strand binding protein, 3 '-5' exonuclease, dNTP and water.
In a third aspect of the present disclosure, a kit for detecting hepatitis a and hepatitis e virus by recombinase polymerase amplification is provided, wherein the kit comprises the primer probe set of the first aspect of the present disclosure.
The invention has the beneficial effects that:
the dual detection method for detecting hepatitis A and hepatitis E through the recombinase polymerase isothermal amplification technology, which is established by the method, can realize rapid, comprehensive, sensitive, specific and automatic result judgment which cannot be completed by morphology, immunology, LAMP and single real-time fluorescence detection, and achieves the following detection effects:
short time
Reverse transcription recombinase polymerase amplification technology (RT-RPA) can directly use RNA as a template to realize the integration of reverse transcription and a detection process by only mixing an RPA system with reverse transcriptase to construct an RT-RPA reaction system without converting the RNA into cDNA and then carrying out amplification reaction. The whole reaction can be completed within 20min, while the reverse transcription process of the technologies such as RT-PCR, Real-time PCR, LAMP and the like only needs 30min, and the whole reaction can be completed within 60-90 min.
(II) the requirement on the instrument platform is low
The RPA reaction can be carried out at normal temperature without a special thermal cycle amplification instrument, and the on-site emergency detection of hepatitis A and hepatitis E is better realized.
(III) good specificity
The RPA can identify the SNP of the primer binding region, so that the SNP has strong identification capability to non-detection targets, while LAMP and common Real-time PCR can not identify the SNP, so that the specificity of the RPA detection is obviously superior to that of LAMP and Real-time PCR. The specificity of the detection method established by the invention is also embodied in the specificity of a whole set of primer probes. All primer probes are subjected to Blast comparison analysis, and have high conservation and specificity; meanwhile, the nucleic acids including hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus and the like can be well distinguished through specific experiment verification, and the detection method is proved to have high specificity and can accurately distinguish non-detection targets.
(IV) minimum detection Limit
The lowest detection limit of the detection method established by the invention can reach 10 copies/reaction.
(V) the cost is lower
The method for detecting hepatitis A and hepatitis E by the recombinase polymerase isothermal amplification technology reduces the labor cost and the time cost in operability. The method does not need a complex high-end instrument, the reaction condition can be only one step of 39 ℃, and complex operation is not needed.
(VI) prevention of false negative results
The IAC added in the reaction system can effectively prompt false negative detection results caused by misoperation, reaction inhibitors and the like.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
One embodiment of the present disclosure provides a primer probe set for detecting hepatitis a and hepatitis e viruses by recombinase polymerase amplification, wherein the primer probe set comprises a primer having a nucleotide sequence represented by SEQ ID nos. 1 to 4, and a probe having a nucleotide sequence represented by SEQ ID nos. 5 to 6;
wherein, in the nucleotide sequence shown in SEQ ID NO.5, the 31 st base from the 5' end is marked with FAM luminous group, the 32 nd base is connected with abasic site, and the 34 th base is marked with quenching group BHQ 1; in the nucleotide sequence shown in SEQ ID NO.6, the 31 st base from the 5' end is marked with a VIC luminescent group, the 32 nd base is connected with an abasic site, and the 34 th base is marked with a quenching group BHQ 1.
The primer probe set disclosed by the invention selects specific detection genes or conserved sequences, and needs to ensure that the primer probe segments can respectively and comprehensively cover hepatitis A virus and hepatitis E virus. In addition, the problem of co-amplification of primer probes of different target genes in a reaction system is fully considered in the design process, so that the consistency of Tm values and the homogenization of GC content are comprehensively considered in the design of the primer probes, the conditions of hairpin structures, primer dimers and the like are avoided as far as possible, and as the RPA has the requirement on the length of the primer of 30-38nt and the longest probe of 45nt, the sequence easily generates a large amount of primer dimers under the constant temperature condition to influence the experimental effect, so that higher requirements are provided for the design of the primer, and the probability of simultaneous amplification of different primer probes in the later period is ensured. Finally, a specific primer probe sequence provided by the disclosure is obtained (see table 1 for details).
TABLE 1 summary of hepatitis A and hepatitis E Virus RPA detection primer probes
The nucleotide sequences of the upstream and downstream primers and the probe for detecting the hepatitis A virus are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 5.
The nucleotide sequences of the upstream and downstream primers and the probe for detecting the hepatitis B virus are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 6.
The nucleotide sequences of the upstream and downstream primers and the probe for detecting the positive internal control are respectively shown by SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9. Wherein, in the nucleotide sequence shown in SEQ ID NO.9, the 19 th base from the 5 'end is marked with a CY5 luminescent group, the 22 th base is connected with an abasic site, the 25 th base is marked with a quenching group BHQ3, and the 3' end is marked with a phosphate group.
In one embodiment of the present disclosure, the present disclosure relates to a method for detecting hepatitis a and hepatitis e virus by Recombinase Polymerase Amplification (RPA), wherein the method comprises the following steps:
(1) extracting total RNA of a sample to be detected;
(2) performing recombinase polymerase amplification reaction by using the total RNA as a template and using the primer probe set disclosed by the disclosure;
(3) collecting fluorescence signals of FAM and VIC detection channels to obtain detection results;
a) if the FAM fluorescence channel has an amplification curve, judging that the sample contains hepatitis A virus;
b) if the VIC fluorescence channel has an amplification curve, the sample is judged to contain the hepatitis E virus.
c) And if the fluorescence channel of the endoplasmic control sample probe has no obvious amplification curve, judging to be non-specific amplification.
In one embodiment of the disclosure, the primer probes for performing the detection of the endoplasmic control sample are primers having a nucleotide sequence shown in SEQ ID NO.7-8, and a probe having a nucleotide sequence shown in SEQ ID NO. 9; in addition, in the nucleotide sequence shown in SEQ ID NO.9, a 19 th base from the 5 'end is marked with a CY5 luminescent group, a 22 th base is connected with an abasic site, a 25 th base is marked with a quenching group BHQ3, and the 3' end is marked with a phosphate group. And judging whether the amplification is non-specific amplification or not according to the existence or nonexistence of the CY5 fluorescence channel amplification curve.
The RPA primer probe concentration and the reaction system are configured as follows:
to a 0.2mL Twist AmpEx reaction tube containing lyophilized enzyme powder (cat # TAEXO02KIT), 29.5. mu.L of rehydration buffer, 2.5. mu.L (280mmol/L) of magnesium acetate solution, 1.5 to 3. mu.L (10. mu.M) of each primer, 0.2 to 1. mu.L (10. mu.M) of probe, 1. mu.L (200U/. mu.L) of reverse transcriptase, 5. mu.L of template were added to total 50. mu.L, and the remainder was made up with water. Each primer was used at a final concentration of 3.5 to 4.5. mu.M, and each probe was used at a final concentration of 0.8 to 1.2. mu.M.
In one embodiment of the disclosure, the conditions of the RPA reaction comprise isothermal amplification at 25-43 ℃ for 5-20 min. In a particularly preferred embodiment of the present disclosure, to improve the sensitivity and specificity of the reaction, the conditions of the RPA reaction include isothermal amplification at 39 ℃ for 20 min.
The direct purpose of the detection method of the present disclosure is not to obtain diagnostic results and health conditions, but to detect only samples that have been detached from the human or animal body, and to obtain only information as intermediate results.
The disclosure also provides a kit containing the RPA detection primer probe group.
Preferably, the kit also contains an internal quality control sequence template.
In one embodiment of the present disclosure, the kit further comprises 5 × reaction system buffer, lyophilized recombinase powder, lyophilized polymerase powder, reverse transcriptase, 10 × primer-probe mixture, positive control, and ultrapure water.
Hereinafter, the present disclosure will be described in further detail by examples.
Example 1
This example illustrates the hepatitis A and hepatitis E virus specific primer probe validation assays.
(1) Synthesis of primers and probes
The company, committed to reagents, synthesized the primer probe sets 1 to 3 shown in Table 2.
TABLE 2
Example 2 specificity verification:
the following samples were selected as simulated interference samples: norovirus (from national CDC, 2001), hepatitis b virus (from national CDC, 2005), hepatitis c virus (from national CDC, 2006), salmonella (from national collection of strains, CVCC3949), shigella (from national collection of strains, CVCC3914), staphylococcus aureus (from national collection of strains, CVCC1882), bacillus cereus (from national collection of strains, CVCC1782), listeria monocytogenes (from national collection of strains, CVCC1345), yersinia enterocolitica (from national collection of strains, CVCC1365), enterobacter sakazakii (from national collection of strains, CVCC1768), escherichia coli (from national collection of strains, CVCC2356), vibrio cholerae (from national collection of strains, CVCC3467), escherichia coli O157 (from national collection of strains, CVCC7389), Nucleic acid of Aeromonas hydrophila (from national center for culture Collection, CVCC4002) was used for specificity evaluation, and the total nucleic acid concentration of each sample was 0.001 ng/. mu.L, and the samples were mixed as a specificity detection template to perform RPA reaction using the primer probe combination shown in Table 1.
Preparing an RPA reaction system: to a total of 50. mu.L of the system, 29.5. mu.L of rehydration buffer, 2.5. mu.L (280mmol/L) of magnesium acetate solution, 2. mu.L (10. mu.M) of each primer, 0.5. mu.L (10. mu.M) of probe, 1. mu.L (200U/. mu.L) of reverse transcriptase, 5. mu.L of template, and the balance of water were added to a 0.2mL twist AmpEx reaction tube (cat # TAEXO02KIT) containing lyophilized enzyme powder;
reaction conditions for RPA reaction: FAM, VIC and CY5 were chosen as reporter groups and the RPA reaction procedure was as follows: 39 ℃ 10s, 39 ℃ 10s, 39 ℃ 10s (fluorescence collected), 40 cycles.
Judging the RPA reaction result: blank control, IAC and negative control are established, otherwise, the result of the experiment is invalid; if the FAM channel has an amplification curve, the primer pair of SEQ ID NO.1-2 and the probe of SEQ ID NO.5 have non-specific amplification; if the VIC channel has an amplification curve, the primer pairs of SEQ ID NO.3-4 and the probe of SEQ ID NO.6 have non-specific amplification. Non-specific amplification did not occur in both FAM and VIC channels.
Example 3 verification of minimum detection limit:
the use concentration is 104The copied/mu L of hepatitis A virus nucleic acid and hepatitis E virus nucleic acid are mixed in equal proportion as template, and are diluted to 10 degree by gradient3Copy/. mu.L, 102Copy/. mu.L, 10 copies/. mu. L, L, and 1 copy/. mu.L. The verification was carried out according to the reaction procedure of the above reaction system. The system preparation and the RPA reaction conditions are carried out simultaneouslyExample 2 evaluation of specificity. So that the concentrations of the viruses in the reaction system were 5X 10, respectively4Copy/system, 5X 103Copy/system, 5X 102Copy/system, 50 copy/system, 10 copy/. mu.L and 5 copy/system.
Judging the RPA reaction result: blank control, IAC and negative control are established, otherwise, the result of the experiment is invalid; if the FAM channel has an amplification curve, the primer pair of SEQ ID NO.1-2 and the probe of SEQ ID NO.5 can detect the template with the concentration; if the VIC channel has an amplification curve, the primer pairs of SEQ ID NO.3-4 and the probe of SEQ ID NO.5 can detect the template at that concentration.
The results show that the lowest detection limit of the kit for the target reaches 10 copies/system (2 copies/. mu.L).
Example 4 sample tolerance testing
RNA of hepatitis A virus and hepatitis E virus is extracted by ethanol precipitation method as template, and amplification is carried out according to the reaction system and the reaction program.
Judging the RPA reaction result: blank control, IAC and negative control are established, otherwise, the result of the experiment is invalid; if the FAM channel has an amplification curve, the primer pair of SEQ ID NO.1-2 and the probe of SEQ ID NO.3 can detect the template with the concentration; if the VIC channel has an amplification curve, the primer pairs of SEQ ID NO.4-5 and the probe of SEQ ID NO.6 can detect the template at that concentration.
The result shows that the kit can obtain positive results by amplifying the template simply extracted by ethanol.
Example 5 analysis of coverage
10 strains of hepatitis A Virus and hepatitis E Virus were selected as templates for coverage evaluation, respectively, and amplification was carried out according to the above reaction system and reaction procedure at a nucleic acid concentration of 0.001 ng/. mu.L.
Judging the RPA reaction result: blank control, IAC and negative control are established, otherwise, the result of the experiment is invalid; if the FAM channel has an amplification curve, the primer pair of SEQ ID NO.1-2 and the probe of SEQ ID NO.5 can detect the template; if the VIC channel has an amplification curve, the primer pair of SEQ ID NO.3-4 and the probe of SEQ ID NO.6 can detect the template.
The result shows that the kit can carry out coverage detection on 20 hepatitis A viruses and 20 hepatitis E viruses.
EXAMPLE 6 shelf-Life testing of kits
The RNA of hepatitis A virus and hepatitis E virus was used as a template for evaluation at 100 copies/. mu.L.
On day 0, 10 portions were frozen in a-70 ℃ refrigerator. And (3) storing the assembled kit at the temperature of-20 ℃, and performing storage period tests on the kit with the time periods of 0, 10, 15, 30, 60, 90, 120, 150, 180 and 360 days respectively. The results of the shelf life measurements are shown in table 3.
TABLE 3 shelf life test results
| Shelf life | Hepatitis A virus | Hepatitis E virus |
| Day 0 | + | + |
| Day 10 | + | + |
| Day 15 | + | + |
| Day 30 | + | + |
| Day 60 | + | + |
| Day 90 | + | + |
| Day 120 | + | + |
| Day 150 | + | + |
| Day 180 | + | + |
| Day 360 | + | + |
As can be seen from Table 3, the kit is stored in a refrigerator at the temperature of 20 ℃ below zero, the detection is positive in different storage periods, and the experimental result shows that the storage period of the kit is at least one year.
Comparative example 1
(1) Primer and probe synthesis
Primers and probes for RT-RPA detection of hepatitis A Virus and hepatitis E Virus were synthesized according to the sequences shown in Table 4.
TABLE 4 summary of probes for comparative primers for detecting hepatitis A Virus and hepatitis B Virus
(2) Specificity verification
The following samples were selected as simulated interference samples: norovirus (from national CDC, 2001), hepatitis b virus (from national CDC, 2005), hepatitis c virus (from national CDC, 2006), salmonella (from national collection of strains, CVCC3949), shigella (from national collection of strains, CVCC3914), staphylococcus aureus (from national collection of strains, CVCC1882), bacillus cereus (from national collection of strains, CVCC1782), listeria monocytogenes (from national collection of strains, CVCC1345), yersinia enterocolitica (from national collection of strains, CVCC1365), enterobacter sakazakii (from national collection of strains, CVCC1768), escherichia coli (from national collection of strains, CVCC2356), vibrio cholerae (from national collection of strains, CVCC3467), escherichia coli O157 (from national collection of strains, CVCC7389), Nucleic acid of Aeromonas hydrophila (from national culture Collection, CVCC4002) was used for specificity evaluation, and the total nucleic acid concentration of each sample was 0.001 ng/. mu.L, and the samples were mixed as a specificity detection template for amplification of RT-RPA.
The reaction system preparation and reaction procedure were the same as in example 2.
The results showed that the above reaction results were all negative, and the primers and probes in the comparative examples had good specificity.
(3) Minimum detection limit verification
The use concentration is 104The copied/mu L of hepatitis A virus nucleic acid and hepatitis E virus nucleic acid are mixed in equal proportion as template, and are diluted to 10 degree by gradient3Copy/. mu.L, 102Copy/. mu.L, 10 copies/. mu.L, 2 copies/. mu.L, and 1 copy/. mu.L. The verification was carried out according to the reaction procedure of the above reaction system. The system formulation and RPA reaction conditions were the same as in example 2. So that the concentrations of the viruses in the reaction system were 5X 10, respectively4Copy/system, 5X 103Copy/system, 5X 102Copy/system, 50 copy/system, 10 copy/. mu.L and 5 copy/system.
And (3) judging the RT-RPA reaction result: blank control, IAC and negative control are established, otherwise, the result of the experiment is invalid; if the FAM channel has an amplification curve, the primer pair of SEQ ID NO.10-11 and the probe of SEQ ID NO.12 can detect the template at the concentration; if the VIC channel has an amplification curve, the primer pair of SEQ ID Nos. 13-14 and the probe of SEQ ID No.15 can detect the template at that concentration.
The results show that the lowest visible detection limit for hepatitis A Virus and hepatitis E Virus in the comparative examples is 100 copies/system.
(4) Coverage verification
10 strains of hepatitis A Virus and hepatitis E Virus were selected as templates for coverage evaluation, respectively, and amplification of RT-RPAR was carried out according to the above reaction system and reaction procedure at a nucleic acid concentration of 0.001 ng/. mu.L.
And (3) judging the RT-RPA reaction result: if the FAM channel has an amplification curve, the primer pair of SEQ ID NO.10-11 and the probe of SEQ ID NO.12 can detect the template; if the VIC channel has an amplification curve, the primer pair of SEQ ID NO.13-14 and the probe of SEQ ID NO.15 can detect the template.
The results show that the comparative example protocol missed 1 hepatitis A Virus and 2 hepatitis E viruses.
As can be seen from the comparison of the examples and the comparative examples, the detection method established by the invention can detect the hepatitis A virus and the hepatitis E virus at one time, the detection can be completed in only 20min, while the comparative example needs 90 min; the kit can identify the mononucleotide mutation points in the primer binding region and has strong identification capability for non-detection purposes; the kit has high specificity, can accurately distinguish non-detection targets, and has simple and reliable structure judgment; the kit has strong detection capability on trace nucleic acid in a sample.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
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<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 (6)
1. The primer probe group for detecting the hepatitis A and the hepatitis E viruses by recombinase polymerase amplification is characterized by consisting of a primer for detecting the hepatitis A viruses shown by SEQ ID NO.1-2, a primer for detecting the hepatitis E viruses shown by SEQ ID NO.3-4, a probe for detecting the hepatitis A viruses shown by SEQ ID NO.5, a probe for detecting the hepatitis E viruses shown by SEQ ID NO.6, upstream and downstream primers for detecting positive internal controls shown by SEQ ID NO.7-8 and a probe for detecting positive internal controls shown by SEQ ID NO. 9;
wherein, in the nucleotide sequence shown in SEQ ID NO.5, the 31 st base from the 5' end is marked with FAM luminous group, the 32 nd base is connected with abasic site, and the 34 th base is marked with quenching group BHQ 1; in the nucleotide sequence shown in SEQ ID NO.6, a VIC luminescent group is marked at the 31 st base from the 5' end, an abasic site is connected behind the 32 nd base, and a quenching group BHQ1 is marked at the 34 th base; in the nucleotide sequence shown in SEQ ID NO.9, a 19 th base from the 5 'end is marked with a CY5 luminescent group, a 22 th base is connected with an abasic site, a 25 th base is marked with a quenching group BHQ3, and the 3' end is marked with a phosphate group.
2. A method for the detection of hepatitis a and hepatitis e viruses using recombinase polymerase amplification for non-diagnostic purposes, said method comprising the steps of:
(1) extracting total RNA of a sample to be detected;
(2) performing a recombinase polymerase amplification reaction using the total RNA as a template and the primer probe set of claim 1;
(3) collecting fluorescence signals of FAM and VIC detection channels to obtain detection results;
a) if the FAM fluorescence channel has an amplification curve, judging that the sample contains hepatitis A virus;
b) if the VIC fluorescence channel has an amplification curve, the sample is judged to contain the hepatitis E virus.
3. The method of claim 2, wherein the final use concentration of each primer is 3.5-4.5 μ M and the final use concentration of each probe is 0.8-1.2 μ M.
4. The method of claim 2 or 3, wherein the conditions for the recombinase polymerase amplification reaction comprise isothermal amplification at 25-43 ℃ for 5-20 min.
5. The method of claim 4, wherein the amplification reaction system comprises a positive internal control, a reverse transcriptase, a reaction system buffer, a DNA recombinase, a DNA polymerase, a single-strand binding protein, a 3 '-5' exonuclease, dNTPs, and water.
6. A kit for detecting hepatitis a and hepatitis e viruses by recombinase polymerase amplification, said kit comprising the primer probe set of claim 1.
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| CN108841926B (en) * | 2018-07-13 | 2021-10-01 | 锦州医科大学 | Primer, probe and kit for dual detection of hepatitis E virus and hepatitis A virus by RT-RPA-lateral flow chromatography |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296677A (en) * | 2015-12-08 | 2016-02-03 | 湖南圣湘生物科技有限公司 | Fluorescence quantitative PCR detection kit for hepatitis A viruses and application method thereof |
| CN105296676A (en) * | 2015-12-08 | 2016-02-03 | 湖南圣湘生物科技有限公司 | Fluorescent quantitative PCR detecting kit for hepatitis E virus and using method of fluorescent quantitative PCR detecting kit |
| CN106435026A (en) * | 2016-10-13 | 2017-02-22 | 山东省疾病预防控制中心 | Primer set, probe and test kit for detection of enteroviruses |
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| EP2290096B1 (en) * | 2002-02-21 | 2014-11-19 | Alere San Diego, Inc. | Recombinase polymerase amplification using a temperature-sensitive recombinase agent |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296677A (en) * | 2015-12-08 | 2016-02-03 | 湖南圣湘生物科技有限公司 | Fluorescence quantitative PCR detection kit for hepatitis A viruses and application method thereof |
| CN105296676A (en) * | 2015-12-08 | 2016-02-03 | 湖南圣湘生物科技有限公司 | Fluorescent quantitative PCR detecting kit for hepatitis E virus and using method of fluorescent quantitative PCR detecting kit |
| CN106435026A (en) * | 2016-10-13 | 2017-02-22 | 山东省疾病预防控制中心 | Primer set, probe and test kit for detection of enteroviruses |
Non-Patent Citations (2)
| Title |
|---|
| Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis;Hosam Zaghloul 等;《World Journal of Hepatology》;20141227;第6卷(第12期);摘要、第918页右栏最后1段至第919页右栏倒数第3段、结论部分、图1-2 * |
| 乙型肝炎病毒环介导等温扩增检测法的建立;邵靓婧 等;《医学研究生学报》;20161208;第29卷(第12期);第1309-1314页 * |
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