AU2021102539A4

AU2021102539A4 - A primer, probe and kit for dual detection of hepatitis E virus and hepatitis A virus by rt-rpa-side flow chromatography

Info

Publication number: AU2021102539A4
Application number: AU2021102539A
Authority: AU
Inventors: Wenlong Duan; Shenyang Gao; Dandan Li; Hui Li; Zongshuai Ling; Jiaying Wang; Naifu WANG; Shude Wang; Zhenzhu Weng; Enhui Zha; Tiezhong Zhou
Original assignee: Jinzhou Medical University
Current assignee: Jinzhou Medical University
Priority date: 2021-05-13
Filing date: 2021-05-13
Publication date: 2021-07-01
Anticipated expiration: 2029-05-13

Abstract

The present invention discloses a kit for simultaneously detecting Hepatitis A Virus and Hepatitis E Virus by RT-RPA-lateral flow chromatography, including: a forward primer, a middle probe and a reverse primer of a sequence of an ORF2 gene of Hepatitis E Virus, and/or a forward primer, a middle probe and a reverse primer of a sequence of a VP1 gene of Hepatitis A Virus, which are applied to an RT-RPA amplification technology, conventional reagents required by a recombinase polymerase amplification technology, and a lateral flow chromatography dipstick, wherein the lateral flow chromatography dipstick includes a sampling pad, a control line, a No. 1 detection line and/or a No. 2 detection line. By cooperation of the control line, the detection lines and the primers as well as labels of the probes, the kit can quickly, sensitively, specifically detect the ORF2 gene of Hepatitis E Virus and the VP1 gene of Hepatitis A Virus in a detection sample on site in a non-experimental environment. Drawings ........... 12 C 1.HEV-HAV Double positive 12C 2. HEV Single positive 12C 3. HAV Single positive 12 C 4.HEV-HAV Double negative Fig. 3

Description

Drawings

.... 12 C 1.HEV-HAV Double positive

12C 2. HEV Single positive

12C 3. HAV Single positive

12 C 4.HEV-HAV Double negative

Fig. 3

Description Primer, Probe and Kit for Simultaneously Detecting Hepatitis E Virus and Hepatitis A Virus

by RT-RPA-Lateral Flow Chromatography

Technical Field

The present invention belongs to the technical field of biological nucleic acid molecular

detection, and particularly relates to a method and a kit for simultaneously detecting Hepatitis E

Virus and Hepatitis A Virus by using a combination of Reverse Transcript Recombinase

Polymerase Amplification (RT-RPA) and a lateral flow chromatography dipstick.

Background Art

China has a large burden of liver diseases. Besides Hepatitis B, Hepatitis A and Hepatitis E

are two important diseases that have the highest incidence and spread via the digestive tract.

According to clinical and epidemiological observations, Hepatitis A Virus (HAV) mostly affects

children and young people. The incidence decreases with age. The clinical manifestations usually

start with fever, fatigue and inappetence, followed by hepatomegaly, tenderness, and liver

impairment. Some patients may develop jaundice. Hepatitis E Virus (HEV) mostly affects young

adults over the age of 20, causing hepatitis that is very similar to HAV in clinical symptoms, but it

mostly causes an acute outbreak, and especially in pregnant women infected during the pregnancy

period, the fatality rate is more than 40%. Both HAV and HEV can be excreted with the patient's

feces. Dissemination via polluted water sources, food, seafood (such as scapharca subcrenata),

tableware, etc., can cause a sporadic epidemic or a pandemic. They can also spread via blood

transfusion or injection. In particular, HEV is zoonotic and distributed globally. The nucleic acid

homology of human and pig-derived HEV can be more than 99%. Therefore, HAV and HEV are

listed by the WHO as important public health problems in developing countries.

At present, the detection for Hepatitis A and E is mostly implemented by using an IgM

antibody capture ELISA and a gold immunochromatography assay (GICA) technology, but due to

complicated operation procedures and a long time of ELISA, it brings a lot of inconvenience to

the laboratory. Although a one-step ELISA method proposed in the late period simplifies the

operation procedures and shortens the detection time, it brings the problem that some strongly

positive specimens will be positive. The gold immunochromatography assay (GICA) technology

has the problems such as great difficulty in control of colloidal gold and long time needed for

Description weakly positive specimens; and the above-mentioned methods have defects of high detection costs and inconvenience in operation, which are not conducive to the application of rapid on-site detection. Therefore, it is urgent to develop a rapid, highly sensitive and specific, reliable novel method which can be used in clinical on-site detection. Recombinase polymerase amplification (RPA) technology is a nucleic acid detection technology developed by the British company TwistDx Inc, which is different from PCR. It is a novel rapid isothermal amplification technology for nucleic acid molecules and also known as a nucleic acid detection technology that can replace PCR. The technology mainly includes: at a constant temperature of 37°C, a recombinase can bind tightly to DNA of a primer to form a polymer of the enzyme and the primer. When the primerfinds a completely complementary sequence on a template DNA, the template DNA is melted with the help of single-stranded DNA binding (SSB) protein, and under the action of DNA polymerase, a new DNA complementary strand is formed. A reaction product also increases exponentially, and an amplified fragment that can be detected by agarose gel electrophoresis can be usually obtained within 1 hour. In addition, a reverse primer labeled with FAM or DIG molecule and a probe labeled with 5'biotin are added to an RPA amplification system, an abasic site (dSpacer) or THF is modified at about a 30th base of a fluorescent group labeled by the probe, and this site is a substrate on which a DNA repairing enzyme acts, and can be recognized and excised by ribozyme NFO to produce a new 3' hydroxyl terminal, and then continue to replicate and extend under the action of DNA polymerase, so that a double labeled signal is synchronized with the accumulation of the amplified product, and a detection result can be specifically shown on a lateral flow chromatography dipstick of colloidal gold (GICA) or colloidal carbon (CCICA) which is anti-biotin gold label binding and anti-antibody capturing by molecule marker. The whole reaction is simple and fast, and because there is no need for high temperature cycling, it is especially suitable for use in non-laboratory detection places with a large number of samples. However, at present there is no report on the application of the RPA technology in HAV or HEV detection both in China and abroad.

Summary of the Invention Aiming at the existing problems and status of the prior art, the present invention applies an RPA technology to HAV and HEV detection for the first time, and provides a primer pair and a probe as well as a kit for simultaneously detecting Hepatitis E Virus and Hepatitis A Virus by

Description using RT-RPA-lateral flow chromatography. The method can accurately, fast, and easily detect

RPA of HAV and/or HEV at the same time by crude extraction of virus RNA from a clinical

sample and RT-RPA isothermal amplification on the RNA, and results can be clearly shown on a

lateral flow chromatography dipstick without a need for amplification in a PCR apparatus. It has

advantages of high sensitivity, strong specificity, simple operation procedures and short detection

time, is suitable for clinical on-site detection in a non-laboratory environment, and has a wide

application prospect.

In order to solve the above technical problems, the present invention adopts the following

technical solutions:

the present invention provides a set of a primer pair and a probe for simultaneously detecting

Hepatitis E Virus and Hepatitis A Virus by using RT-RPA-lateral flow chromatography, which

specifically are the primer pair and the probe for detecting an ORF2 gene of Hepatitis E Virus

and/or a VP1 gene of Hepatitis A Virus, wherein a sequence of a forward primer of the ORF2

gene of Hepatitis E Virus is shown as SEQ ID No. 1, a sequence of a reverse primer is shown as

SEQ ID No. 2, and sequences of a probe are shown as SEQ ID No. 3 and SEQ ID No. 4; a

sequence of a forward primer of the VP1 gene of Hepatitis A Virus is shown as SEQ ID No. 5, a

sequence of a reverse primer is shown as SEQ ID No. 6, and sequences of a probe are shown as

SEQ ID No. 7 and SEQ ID No. 8.

for the probe of the ORF2 gene of Hepatitis E Virus, a 5' terminal of the sequence shown as

SEQ ID No. 3 is connected with a first labeled molecule, a 3' terminal of the sequence shown as

SEQ ID No. 4 is connected with a protection label for preventing polymerisation, and a 3'

terminal of the sequence shown as SEQ ID No. 3 and a 5'terminal of the sequence shown as SEQ

ID No. 4 are connected by an artificial base analogue that can be recognized and excised by an

enzyme having DNA damage repairing activity;

for the probe of the VP1 gene of Hepatitis A Virus, a 5' terminal of the sequence shown as

SEQ ID No. 7 is connected with a first labeled molecule, a 3' terminal of the sequence shown as

SEQ ID No. 8 is connected with a protection label for preventing polymerisation, and a 3'

terminal of the sequence shown as SEQ ID No. 7 and a 5'terminal of the sequence shown as SEQ

ID No. 8 are connected by an artificial base analogue that can be recognized and excised by an

enzyme having DNA damage repairing activity;

a 5' terminal of the reverse primer of the ORF2 gene of Hepatitis E Virus is connected with a

Description second labeled molecule; and a 5' terminal of the reverse primer of the VP1 gene of Hepatitis A Virus is connected with a third labeled molecule. The probe and the forward primer of the ORF2 gene of Hepatitis E Virus are in the same amplification direction; and the probe and the forward primer of the VP1 gene of Hepatitis A Virus are in the same amplification direction. Preferably, in the above-mentioned probes, the protection label for preventing polymerisation is C3-spacer; and the artificial base analogue which can be recognized and excised by the enzyme having DNA damage repairing activity is tetrahydrofuran (THF). Preferably, the first labeled molecule is biotin (Biotin), the second labeled molecule is a fluorescent molecule (FAM), and the third labeled molecule is dig (Dig). The primers and the probes of the present invention are selected from HEV or HAV characteristic fragments, designed according to characteristics of HEV or HAV, synthesized by using an artificial chemical method and added with a DNA fingerprint sequence of a specific size and a molecule marker. An application of the above-mentioned sets of the primer pairs and the probes in detecting Hepatitis E Virus and/or Hepatitis A Virus reagents. The present invention further provides a kit for simultaneously detecting Hepatitis E Virus and Hepatitis A Virus by using RT-RPA-lateral flow chromatography, including the above-mentioned sets of the primer pairs and the probes. Preferably, the kit further includes a lateral flow chromatography dipstick, wherein the lateral flow chromatography dipstick includes a sampling pad, a control line, a No. 1 detection line and/or a No. 2 detection line. Further, preferably, the sampling pad contains a specific conjugate of a first labeled molecule labeled by colloidal carbon particles; further, preferably, the control line is coated with the specific conjugate of the first labeled molecule; further, preferably, the No. 1 detection line is coated with a specific conjugate of a second labeled molecule; further, preferably, the No. 2 detection line is coated with a specific conjugate of a third labeled molecule; and

Description further, preferably, the specific conjugate of the first labeled molecule is streptavidin; the

specific conjugate of the second labeled molecule is an FAM antibody; and the specific conjugate

of the third labeled molecule is a dig antibody.

The present invention further provides a method for simultaneously detecting Hepatitis E

Virus and Hepatitis A Virus by using RT-RPA-lateral flow chromatography, wherein the method

includes: amplifying a sequence of an ORF2 gene of Hepatitis E Virus and/or a sequence of a VP1

gene of Hepatitis A Virus by using an RAP technology, and displaying an amplified product with

a lateral flow chromatography dipstick.

Specifically, the method includes the following steps:

(1) extracting total RNA of a sample to be detected by means of a paramagnetic particle

method-based virus RNA extraction kit;

(2) reversely transcribing the RNA to obtain cDNA; and performing RPA amplification on

the obtained cDNA by using the above-mentioned primers and probes of the ORF2 gene of

Hepatitis E Virus and/or the VP1 gene of Hepatitis A Virus; and

(3) detecting an amplified product by use of a lateral flow chromatography dipstick, and

determining whether the sample contains Hepatitis E Virus and/or Hepatitis A Virus;

wherein a determination method includes: in 10 minutes, (1) determining that the experiment

is successful when a control C line appears, and determining that HEV and HAV simultaneously

exit and detection results both are positive when No. 1 and No. 2 detection lines simultaneously

appear; (2) determining that the experiment is successful when the control C line appears, and

determining that the HEV detection result is positive and the HAV detection result is negative

when only the No. 1 detection line appears and the No. 2 detection line does not appear; (3)

determining that the experiment is successful when the control C line appears, and determining

that the HAV detection result is positive and the HEV detection result is negative when only the

No. 2 detection line appears and the No. 1 detection line does not appear; (4) determining that the

experiment is successful when the control C line appears, and determining that the HEV and HAV

detection results both are negative when neither of the No. 1 and No. 2 detection lines appears;

and (5) determining the experiment fails and the result is meaningless when the control C line

does not appear.

Preferably, a reverse transcription process in step (2) involves: uniformly mixing a reverse

transcription reaction liquid FastQant with the RNA obtained at step (1), and processing the

Description mixture in a metal bath at a constant temperature of 42°C for 10-15 min.

Further, preferably, a reverse transcription system is of 20 L; wherein a volume ratio of the

reaction liquid to the RNA is 3: 1.

Preferably, an RPA amplification system in step (2) has a total volume of 47.5 L, wherein

the forward primers of the ORF2 gene and the VP1 gene are 2.1 L, respectively, the reverse

primers are 2.1 L, respectively, the probes are 0.6 L, respectively, a Rehydration buffer solution

is 29.5 L, a template cDNA is 5 L, and ddH 20 is 3.4 L.

Preferably, the RPA amplification in step (2) involves: uniformly mixing the RPA

amplification system, adding 2.5 L of magnesium acetate into the amplification system, and

uniformly mixing the amplification system for reaction.

Further, preferably, a reaction condition is as follows: warmly bathing in a metal bath at a

constant temperature of 38°C for 10-15 min.

Preferably, step (3) specifically includes: taking and balancing a colloidal carbon detection

card at a room temperature; diluting an amplified product in step (2) with a buffer solution,

dropwise adding the diluted amplified product into a sample hole of the colloidal carbon detection

card and reading a result.

Further, preferably, the buffer solution is 1% PBS-T.

Applications of the kit and the detection method in detecting Hepatitis E Virus and Hepatitis

A Virus reagents.

It should be noted that an effective design of primers and probes is the most critical link to

determine the success of the present invention. However, the RPA technology is in the initial

research stage, and there is no special design software for primers and probes, nor a large amount

of data to provide a basis for a primer design principle. PCR primers are inapplicable to RPA. At

present, in experiments, it is necessary to design multiple pairs of primers from both terminals of

a target sequence for optimization and screening. The design requirements of the primer for this

technology are extremely strict, and the substitution or increase or decrease of individual bases

will have an important impact on the experimental results. The primers and probes that can be

used for clinical detection can only be screened out after experimental verification and testing.

The following factors become the main influencing factors during design: (1) a primer length is

required to be 30-35 bp (too short primer will seriously affect the activity of the recombinase, a

long primer may not necessarily improve the amplification performance, but also increase the

Description possibility of the formation of a secondary structure), a probe length is required to be 46-52 bp,

and a GC content is 40-60% (3-5 nucleotides at the 5' terminal should avoid the polymerization

with guanine (G) and cytosine (C) so as to promote the binding of the primer and the amplified

target gene. For 3 nucleotides at the 3' terminal, guanine and cytosine contribute to the stable

binding of the polymerase, and can improve the amplification performance of the primer), the

occurrence of a secondary structure inside the primer and the occurrence of repetitive sequences

of the primer are avoided, (2) an amplified fragment for detection is less than 500 bp, (3) when

the primers and the probes are designed, the formation of sequences of a secondary structure,

primer-primer interaction, primer-probe interaction and a hairpin structure should be avoided as

much as possible, and the formation of a dimer should be reduced. Secondly, since the present

application involves the amplification of the primers and the probes of the two genes in the same

system, secondary structures and hairpin structures will appear between the primers and probes of

the two genes, which again increases the difficulty in design of the primer and probe, and

avoiding possible problems between the two also becomes another important factor to be

considered in the primer and probe design of the present invention.

The present invention determines conservative core areas based on comparative analysis of

homologous sequences of HEV ORF2 of DQ279091, AJ272108, AF060668, AF060669, D11092,

M73218, GU206559 and GU119961, and HAV VP1/VP3 of KX088647, KX343015, KX343016,

KX343017 and KX343018 from the GenBank, and overcomes the above-mentioned problems in

the primer design and designs multiple sets of primer pairs and probes for these areas at the same

time to perform RPA amplification screening. Meanwhile, deionized water is used as a template to

amplify the designed primer pairs and probes, and an amplified product is used as a negative

control. Finally, a pair of amplified products of which a detection line and a control line can be

clearly shown in a colloidal carbon detection card (see Fig. 3) are selected.

Experimental principle: NFO-RPA is a reverse amplified product formed by adding an NFO

enzyme (endonuclease IV), a specific molecule labeled nucleic acid probe and a terminal

modification on basis of the RPA amplification system, as shown in Fig. 2, a design principle of

the molecule labeled nucleic acid probe is to label a 5' terminal of the nucleic acid probe with

Biotin (biotin), introduce a heterologous nucleotide residue THF (tetrahydrofuran) in the middle,

and add a blocker C3-spacer to a 3' terminal of the nucleic acid probe to prevent amplifying

non-target DNA by inducting Bsu polymerase. Only after complementation fixation of the probe

Description and a DNA strand, the NFO enzyme having DNA damage repairing activity recognizes and

excises the THF at a specific site, the Bsu polymerase continuously amplify and extent along this

site, and finally forms a specific amplicon product with an forward 5' terminal labeled with a

biotin probe and a reverse 5' terminal labeled with FAM or Dig (dig). Positions, respectively

coated with an FAM antibody, a Dig antibody and Biotin, of a detection area on the lateral flow

chromatography dipstick are the No. 1 detection line, the No. 2 detection line, and the control C

line. After the NFO-RPA amplicon product is dropwise added into a sampling slot, Strep

(streptomycin, Biotin-ligand) labeled with colloidal carbon particles on the sampling pad will be

specifically bound to a 5' Biotin (biotin) molecule of the NFO-RPA amplicon while flowing with

the liquid sample, and meanwhile, the other end of the amplicon will be specifically captured by

the molecules coating the No. 1 line or No. 2 line to form a sandwich complex, Strep

(streptomycin) labeled with the colloidal carbon particles that is not specifically bound to the

amplicon is bound to the Biotin (biotin) coated on the control C line to form a control detection

line.

The beneficial effects of the present invention are as follow:

1. The present invention uses the RPA technology to establish a method for fast and

simultaneously detecting HAV and HEV for the first time, passes evaluation on specificity,

sensitivity and repeatability, can be used for clinical on-site detection, and provides a novel

sensitive and reliable method for HAV and HEV on-site detection. The RPA technology greatly

shortens the detection time and simplifies the reaction procedure. The combination of the RPA

technology and the DNA rapid extraction technology makes field detection possible and has a

wide application prospect.

2. The present invention overcomes the problems in the amplification of two sets of specific

primers and specific labeled probes in the same system, and achieves the goal of simultaneous

identification and detection of HEV and/or HAV.

3. The present invention eliminates an original process of performing gel electrophoresis on

a PCR or LAMP amplified product to determine a result, and the combination of the RPA

amplification and the lateral flow chromatography dipstick allows a detection result to be directly

read on site, thereby realizing on-site and rapid molecular detection of HEV and HAV.

4. The present invention realizes the on-site and rapid molecular detection of HEV and HAV.

The RPA technology is a method for amplifying nucleic acids at a constant temperature, so there

Description is no need for a PCR apparatus, which overcomes a disadvantage of high cost of PCR apparatus;

the RPA technology enable the RPA amplification reaction can be completed within about 20

minutes, and saves a lot of time compared with the PCR method; and the combination of the RPA

method and the lateral flow chromatography dipstick allows the detection result to be directly

read on site without the dependence on an expensive gel imaging system and equipment such as a

computer, so as to realize the on-site and rapid molecular detection of HEV and HAV.

Brief Description of the Drawings

Fig. 1 is a schematic diagram of RPA amplification;

Fig. 2 is a schematic diagram of reading an NFO-RPA amplified product through a lateral flow

chromatography dipstick;

Fig. 3 is a reading and determination method of a dipstick after combining RT-RPA with a lateral

flow chromatography dipstick;

Fig. 4 is a result of a sensitivity test of a dipstick of RT-RPA-lateral flow chromatography

dipstick;

Fig. 5 is a result of a specificity test of a dipstick of RT-RPA lateral flow chromatography dipstick;

Fig. 6 is a result of a repeatability test of an RT-RPA amplification detection method for HEV and

HAV; and

Fig. 7 is a detection result of lateral flow chromatography-RT-RPA for a clinical sample.

Detailed Description of the Invention

In order to better explain the technical solutions of the present invention, embodiments of the

present invention will be described in details below in conjunction with the accompanying

drawings. The embodiments below are used for further illustrating the present invention, but are

not understood as a fixation or limitation for the present invention. Unless otherwise defined, the

technical features can be replaced by other known technical features which have the same or

similar functions or effects in the art without departing from the conception of the present

invention.

Materials and reagents used by the present invention are as follow:

Molecular biological reagents, TwistAmp RT-NFO Kits and lateral flow chromatography

dipsticks were purchased from Twist DX company. Paramagnetic particle method-based virus

Description RNA extraction kits were purchased from Hangzhou Bioer Technology Co., Ltd. Other

biochemical reagents were imported and repackaged or analyzed and purified in China. Primers

and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Apparatus used by the present invention include: a thermostatic metal bath, a mini centrifuge,

a pipettor and so on.

Experimental methods whose specific conditions are not defined in the embodiments were

generally operated according to the conditions in New York: Cold Spring Harbor Laboratory

Press (2001) edited by Sambrook and others, or the conditions suggested by the manufacturers of

the apparatus or reagents.

Embodiment 1: Design and screening of primers and probes

In order to achieve the above-mentioned objects, the present invention designed primers and

probes for HEV ORF2 and HAV VP. Based on the contrastive analysis of homologous

sequences of HEV ORF2 of DQ279091, AJ272108, AF060668, AF060669, D11092, M73218,

GU206559 and GUI1996land HAV VP1/VP3 of KX088647, KX343015, KX343016, KX343017

and KX343018 from the GenBank, conservative core areas of the two genes were determined, and

multiple sets of primers and probes were respectively designed for the areas to screen RAP, and

meanwhile, deionized water was used as a template to amplify the designed primers and probes,

and an amplified product is used as a negative control, finally, a pair of amplified products of

which a detection line and a control line can be clearly shown in a colloidal carbon detection card

(see Fig. 3) are selected. At the end, a set of primers and a probe below was determined as

universal gene detection target sites for detecting HEV and HAV. Forward primer 5'-ATGCCACCATGCGCTCTCGGGCTCTTCTGTT (a sequence is shown as SEQ ID ORF2 gene of No. 1) '

Hepatitis E Reverseprimer 5'-FAM-TGAGAATCAACCCGGTCACCCCAGAA (a sequence is shown as SEQ ID Virus No. 2) ACCAC-3' Molecule labeled probe 5'-Biotin-TCGTGCTTCTGCCTATGCTGCCCGCGC (a partial sequence between Biotin and THF is shown as SEQ ID No. CACC/THF/GCCGGTCAGCCGTCTG-C3 spacer-3' 3, and a partial sequence between THF and C3-spacer is shown as SEQ ID No. 4)

Forward primer 5'-AACAGGTATACAAAGTCAGCACATCAGAAA (a sequence is shown as SEQ ID

Description VP1 gene of No. 5) GGT-3'

Hepatitis A Reverse primer 5'-Dig-CTCCAACTTGTGTAGTAACATCCATAGC (a sequence is shown as SEQ ID Virus No. 6) ATGATAA-3' Molecule labeled probe 5'-Biotin-GCTTATTGTGTATTGTTATAACAGATTG (a partial sequence between Biotin and THF is shown as SEQ ID No. ACC/THF/CTCCTTCTAACGTTGCT-C3 spacer-3' 7, and a partial sequence between THF and C3-spacer is shown as SEQ ID No. 8)

Notes: the 5' terminal of the probe is labeled by biotin, a base at a site which is about 31

bases away from the 5' terminal is substituted by dSpacer, and the 3' terminal is blocked by

C3-spacer.

Biotin: biotin; FAM: fluorophores; dSpacer: abasic site/THF; C3-spacer: polymerase

extending and blocking group

Embodiment 2: Establishment and tests of an RT-RPA amplification method for detecting

HEV and HAV

1. Establishment of the RT-RPA amplification method for detecting HEV and HAV

1.1 Extraction of RNA of HEV and HAV

200 L of serum or saliva of a patient was taken, RNA was extracted by using a

paramagnetic particle method-based virus RNA extraction kit, and washed three times to obtain

100 mg of paramagnetic particles, and finally, the paramagnetic particles were dried and

preserved at a room temperature for later use.

1.2 RT-RPA amplification reaction

Specific sequences of HEV and HAV were amplified by using an RT-RPA method in this

embodiment, and the specific steps are as follow:

(1) 20 L of FastQnat reverse transcription reaction liquid was added into a tube containing

the obtained standby paramagnetic particles and uniformly mixed, the mixture was transferred to

a 0.2 ml reaction tube, and the reaction tube was placed in a metal bath at a constant temperature

of 42°C for processing for 10 min to obtain a cDNA template;

(2) 2.1 L (10 mol/L) of each of the forward primers, 2.1uL (10 mol/L) of each of the

reverse primers, and 0.6 L (10 mol/L) of each of the probes of the ORF2 gene and the VP1

gene, which are designed in Embodiment 1, 29.5 L of a Rehydration buffer solution, and 5 L of

RNA of the cDNA template were added into another 0.2 ml centrifuge tube, ddH20 was added

Description into the centrifuge tube until the volume reached 47.5 L, vertex mixing and short centrifugation

were performed;

(3) 47.5 pL of the mixed solution was transferred into a 0.2 mL TwistAmp NFO reaction tube containing a lyophilized enzyme powder, and the mixture was repeatedly blown and beaten

by a pipettor until the whole particles were dissolved;

(4) 2.5 L (280 mmol/L) of a magnesium acetate solution was added into the reaction tube,

vertex mixing was performed by means of violent up-down reversal, and the reaction occurred

immediately;

(5) short centrifugation was performed, and then the reaction tube was placed in a metal bath

at a constant temperature of 38°C for reaction for 20 min to obtain an RT-RPA reaction product.

1.3 Detection for an amplified product by a CCICD lateral flow chromatography dipstick

A certain number of the lateral flow chromatography dipsticks (PCRD, Abingdon) were

taken for marking different detection samples. 75 L of a hybrid detection buffer solution (PCRD

Buffer) was taken for each detection sample, and added into a reaction tube. 5 L of a reaction

product (the RT-RPA reaction product) was placed into a centrifuge tube and uniformly mixed. A

reaction solution was added in a sample reaction area of the dipstick for reaction at a room

temperature for 5 min, and observation was made immediately. If the reaction area of the dipstick

clearly showed a control line and strip-type No. 1 and No.2 detection lines, it is indicated that

HEV and HAV detection results are both positive; if only the No. 1 or No. 2 detection line

appeared, it is indicated that the HEV or HAV detection result is positive; if neither of the No. 1

and No. 2 detection line appeared, it is indicated that the results are both negative.

2. Tests of the RT-RPA amplification method for detecting HEV and HAV

2.1 Sensitivity test of the RT-RPA amplification method for detecting HEV and HAV

An artificial recombined viral particle fluid of an HEV ORF2-HAV VP1 RNA fragment at a

known concentration of 9.5*105 copies/mL was diluted by 10 times with deionized water, RNA of

the virus was extracted by use of the paramagnetic particle method-based virus RNA extraction

kit, non-eluted paramagnetic particles were used as a RNA carrying template, and the template

was amplified according to the operation method in "1.2 Primer amplification", 5 L of a hybrid

reaction product (the RT-RPA reaction product) was taken and shown on a lateral flow

chromatography dipstick, and a result showed that target molecules (Fig. 4) of 9.5 copies can be

detected, which indicates that the primers designed by the present invention have high sensitivity

Description and accuracy in detecting HEV and HAV, and the operation is simple.

2.2 Specificity test of the RT-RPA amplification method for detecting HEV and HAV

200 L of Norovirus (NoV) at a concentration of 1.6 * 108 copies/mL, Rotavirus (RV) with a

titer of 5.51 gTCID50/mL, Hepatitis C Virus (HCV) at a concentration of 3.5 * 107 copies/mL,

Enterovirus 71 (EV71) at a concentration of 7.5 * 107 copies/mL, Coxsackievirus (CAV) at a

concentration of 5.4 * 107 copies/mL and Poliovirus (PV) with a titer of 5.951 gTCID50/mL were

taken.

The artificial recombined viral particle fluid of the HEV ORF2-HAV VP1 RNA fragment at

the known concentration of 9.5*105 copies/mL was used as a positive control template, and NoV,

RV, HCV, EV71 CAV and PV were amplified by using the primers and probes screened out in

Embodiment 1 according to the isothermal amplification steps in Embodiment 2. Amplified

products were detected by use of the lateral flow chromatography dipsticks. For the HEV

amplified product, the No. 1 and No. 2 detection lines and the control line appeared in the reaction

area of the lateral flow chromatography dipstick, but for the detection samples of other viruses,

only the control line clearly appeared, the detection lines did not appear, and the detection results

were all negative (Fig. 5). It is indicated that the RT-RPA amplification method for simultaneously

detecting HEV-HAV of the present invention has a good specificity.

2.3 Repeatability test of the RT-RPA amplification method for detecting HEV and HAV

the known concentration of 9.5*105 copies/mL was used as a template, and RT-RPA isothermal

amplification was performed according to the primers and the probes screened out in

Embodiments 1 and 2, repetition was made on the template for 3 times, and amplified products

were respectively detected by use of lateral flow chromatography dipsticks. The results showed

that for the amplified products of the templates of the same quantity, the control line and the

detection lines with the same brightness appeared, which indicates that the present invention has a

good repeatability (Fig. 6).

Control 1:

In the present invention, another set of a primer pair and a probe was designed according to

the conservative core areas determined in Embodiment 1 at the same time, which respectively

were a forward primer whose sequence was shown as SEQ ID No. 9 of an ORF gene of Hepatitis

E Virus, a reverse primer whose sequence was shown as SEQ ID No. 10, and a probe whose

Description sequences were shown as SEQ ID No. 11 and SEQ ID No. 12; a forward primer whose sequence was shown as SEQ ID No. 13 of a VP1 gene of Hepatitis A Virus, a reverse primer whose sequence was shown as SEQ ID No. 14, and a probe whose sequences were shown as SEQ ID No. and SEQ ID No. 16. A sensitivity test, a specificity test and a repeatability test for an RT-RPA amplification method for detecting HEA and HAV were performed according to the method in Embodiment 2. Forward primer 5'-CATGCGCTCTCGGGCTCTTCTGTTTCTGTT (a sequence is shown as SEQ ID ORF2 gene of No. 9) -'

Hepatitis E Reverseprimer 5'-FAM-CGAAGGGTTGAGAATCAACCCGGTCA (a sequence is shown as SEQ ID Virus No. 10) CCCCA-3' Molecule labeled probe Biotin-TCTGCCTATGCTGCCCGCGCCACCGGCC (a partial sequence between Biotin and THF is shown as SEQ ID No. GGTCA/THF/CCGTCTGGCCGTCG-C3 spacer-3' 11, and a partial sequence between THF and C3-spacer is shown as SEQ ID No. 12)

Forward primer 5'-AGTGAACAGGTATACAAAGTCAGCACATCA (a sequence is shown as SEQ ID VP1 gene of No. 13) GA-3' Hepatitis A Reverse primer 5'-Dig-CATAGCATGATAAAGAGGAGCAAAACAT (a sequence is shown as SEQ ID Virus No. 14) TCCAA-3' Molecule labeled probe 5'-Biotin-ATTGTGTATTGTTATAACAGATTGACC (a partial sequence between Biotin and THF is shown as SEQ ID No. TCTCCTT/THF/TAACGTTGCTTCCCAT-C3 15, and a partial sequence between spacer-3' THF and C3-spacer is shown as SEQ ID No. 16)

Conclusion: Sensitivity and repeatability of the RT-RPA amplification result based on the sets of the primers and the probes are lower, and specificity is equal to that of the RT-RPA amplification result based on the sets of the primers and the probes of the present invention. It is indicated that the sets of the primers and the probes of the present invention are the best. Embodiment 3: HEV and HAV detection for a clinical sample 1. Lateral flow chromatography RT-RPA for detecting the clinical sample 3 HEV and HAV double positive clinical human serum samples, 7 single positive samples for each of HEV and HAV, and 4 double negative samples, total 21 samples which were detected by an established fluorescent quantitation RT-PCR method for detecting HEV and HAV in the

Description laboratory were taken, and RNA was extracted and amplified according to the RT-RPA detection

method in steps 2.1 and 2.2 of Embodiment 2. By showing an RT-RPA amplified product on a

lateral flow chromatography dipstick, for 3 samples, a control line and No. 1 and No. 2 detection

lines appeared in a reaction area of the dipstick, for 7 samples, the control line and the No. 1

detection line appeared in the reaction area of the dipstick, for 7 samples, the control line and the

No. 2 detection line appeared in the reaction area of the dipstick, and for 4 samples, only the

control line appeared in the reaction area of the dipstick. It is indicated that the coincidence rate of

detection of the RT-RPA method and the fluorescent quantitation RT-PCR method is 100%, and

results are shown in Fig. 7.

Embodiment 4: A kit for detecting HEV-HAV

1. Composition of the kit: the sets of the primers and the probes screened out in Embodiment

1, an HEV-HAV positive control template, a RT reverse transcription liquid, an RPA amplification

Rehydration buffer solution, lyophilized enzyme particles, magnesium acetate (280 mM),

deionized water and lateral flow chromatography dipsticks; and a paramagnetic particle

method-based virus DNA/RNA extraction kit.

2. RT reserve transcription system: 20 [L,

3. RPA amplification system: the amplification system is 50 L, 2.1 L (10 mol/L) of each

of the forward primers, 2.1 L (10 mol/L) of each of the reverse primers, 0.6 L (10 mol/L) of

each of the probes of HEV and HAV, 5 L of the HEV-HAV positive control cDNA template, 29.5

L of the Rehydration buffer solution, 8.2 L of the deionized water and 2.5 L of the magnesium

acetate (280 mM) were added into a 0.2 mL TwistAmp NFO reaction tube containing a

lyophilized enzyme powder.

4. Detection steps

4.1 RT-RPA amplification

RNA was extracted according to the RT-RPA detection method in steps 2.1-2.2 of

Embodiment 2 and reacted with RT, and RPA amplification is performed. That is, coreaction in a

metal bath at a constant temperature of 38C for 25 min.

4.2 Detection by use of a later flow chromatography dipstick

5 L of an RT-RPA amplified product was taken and detected according to the method in

"2.3 Detection of an amplified product by use of a lateral flow chromatography dipstick" of

Embodiment 2, if No. 1 and No. 2 detection lines and a control line appear on the lateral flow

Description chromatography dipstick, HEV and HAV detection results are both positive, if the control line

appears, and only the No. 1 detection line or the No. 2 detection line appears at the same time, the

HEV detection result is positive or the HAV detection result is positive; if only the control line

appears, the detection results are both negative.

Although the specific implementation modes of the present invention are described in details

above in conjunction with the accompanying drawings, the scope of protection of the present

invention is not limited thereto, those skilled in the art should understand that various

modifications and transformations can be made without any creative effort on the basis of the

technical solutions of the present invention, and these modifications and transformations are still

within the scope of protection of the present invention.

Claims

Claims 1. A set of a primer pair and a probe for simultaneously detecting Hepatitis E Virus and

Hepatitis A Virus by using RT-RPA-lateral flow chromatography, characterized by being the

primer pairs and the probes for detecting an ORF2 gene of Hepatitis E Virus and/or a VP1 gene of

Hepatitis A Virus, wherein a sequence of a forward primer of the ORF2 gene of Hepatitis E Virus

is shown as SEQ ID No. 1, a sequence of a reverse primer is shown as SEQ ID No. 2, and

sequences of a probe are shown as SEQ ID No. 3 and SEQ ID No. 4; a sequence of a forward

primer of the VP1 gene of Hepatitis A Virus is shown as SEQ ID No. 5, a sequence of a reverse

primer is shown as SEQ ID No. 6, and sequences of a probe are shown as SEQ ID No. 7 and SEQ

ID No. 8;

for the probe of the ORF2 gene of Hepatitis E Virus, a 5' terminal of the sequence shown as

SEQ ID No. 3 is connected with a first labeled molecule, a 3' terminal of the sequence shown as

SEQ ID No. 4 is connected with a protection label for preventing polymerisation, and a 3'

terminal of the sequence shown as SEQ ID No. 3 and a 5'terminal of the sequence shown as SEQ

ID No. 4 are connected by an artificial base analogue which may be recognized and excised by an

enzyme having DNA damage repairing activity;

for the probe of the VP1 gene of Hepatitis A Virus, a 5' terminal of the sequence shown as

SEQ ID No. 7 is connected with a first labeled molecule, a 3' terminal of the sequence shown as

SEQ ID No. 8 is connected with a protection label for preventing polymerisation, and a 3'

terminal of the sequence shown as SEQ ID No. 7 and a 5'terminal of the sequence shown as SEQ

ID No. 8 are connected by an artificial base analogue which may be recognized and excised by an

enzyme having DNA damage repairing activity;

a 5' terminal of the reverse primer of the ORF2 gene of Hepatitis E Virus is connected with a

second labeled molecule;

a 5' terminal of the reverse primer of the VP1 gene of Hepatitis A Virus is connected with a

third labeled molecule; and

the probe and the forward primer of the ORF2 gene of Hepatitis E Virus are in the same

amplification direction, and the probe and the forward primer of the VP1 gene of Hepatitis A

Virus are in the same amplification direction.
2. The set of the primer pair and the probe of claim 1, characterized in that in the probes, the

protection label for preventing polymerization is C3-spacer; and the artificial base analogue which

may be recognized and excised by the enzyme having DNA damage repairing activity is

Claims tetrahydrofuran (THF); and

the first labeled molecule is biotin (Biotin), the second labeled molecule is a fluorescent

molecule (FAM), and the third labeled molecule is dig (Dig).
3. An application of the set of the primer pair and the probe of claim 1 or 2 in detecting

Hepatitis E Virus and/or Hepatitis A Virus reagents.
4. An kit for simultaneously detecting Hepatitis E Virus and Hepatitis A Virus by using

RT-RPA-lateral flow chromatography, characterized by comprising the sets of the primer pairs and

the probes of claim 1.
5. The kit of claim 4, characterized in that the kit further comprises a lateral flow

chromatography dipstick, wherein the lateral flow chromatography dipstick comprises a sampling

pad, a control line, a No. 1 detection line and/or a No. 2 detection line.
6. The kit of claim 5, characterized in that the sampling pad contains a specific conjugate of

a first labeled molecule labeled by colloidal carbon particles; the control line is coated with the

specific conjugate of the first labeled molecule; the No. 1 detection line is coated with a specific

conjugate of a second labeled molecule; and the No. 2 detection line is coated with a specific

conjugate of a third labeled molecule; and

the specific conjugate of the first labeled molecule is streptavidin; the specific conjugate of

the second labeled molecule is an FAM antibody, and the specific conjugate of the third labeled

molecule is a dig antibody.
7. An application of the kit of claim 5 or 6 in detecting Hepatitis E Virus and/or Hepatitis A

Virus reagents.
8. A method for simultaneously detecting Hepatitis E Virus and Hepatitis A Virus by using

RT-RPA-lateral flow chromatography, characterized by comprising the following steps:

(1) reversely transcribing total RNA extracted from a sample to be detected to obtain cDNA;

performing RPA amplification on the obtained cDNA by using the primers and the probes of the

ORF2 gene of Hepatitis E Virus and/or the VP1 gene of Hepatitis A Virus; and

(2) detecting an amplified product by using a later flow chromatography dipstick, and

determining whether the sample contains Hepatitis E Virus and/or Hepatitis A Virus;

wherein a determination method comprises: in 10 minutes, (1) determining that the

experiment is successful when a control C line appears, and determining that the HEV and the

HAV simultaneously exist and detection results are both positive when No. 1 and No. 2 detection

Claims lines simultaneously appear; (2) determining that the experiment is successful when the control C

line appears, and determining that the HEV detection result is positive and the HAV detection

result is negative when only the No. 1 detection line appears and the No. 2 detection line does not

appear; (3) determining that the experiment is successful when the control C line appears, and

determining that the HAV detection result is positive and the HEV detection result is negative

when only the No. 2 detection line appears and the No. 1 detection line does not appear; (4)

determining that the experiment is successful when the control C line appears, and determining

that neither of the HEV and HAV exist and the detection results both are negative when neither

the No. 1 and No. 2 detection lines appears; and (5) determining the experiment fails and the

detection result is meaningless when the control C line does not appear.
9. The method of claim 8, characterized in that a reverse transcription process in step (1)

comprises: uniformly mixing a reaction liquid FastQant with the RNA, and processing the

mixture in a metal bath at a constant temperature of 42C for 10-15 min;

an RPA amplification system has a total volume of 47.5 L, wherein the forward primers of

the ORF2 gene and the VP1 gene are 2.1 L, respectively, the reverse primers are 2.1 L, respectively, the probes are 0.6 L, respectively, a Rehydration buffer solution is 29.5 L, a

cDNA template is 5 L, and ddH20 is 3.4 L;

the RPA amplification in step (1) comprises: uniformly mixing the RPA amplification system,

adding 2.5 L of magnesium acetate, and uniformly mixing for reaction.

Step (2) specifically comprises: taking and balancing a colloidal carbon detection card;

diluting the amplified product in step (1) with a buffer solution, dropwise adding the diluted

amplified product into a sample hole of the colloidal carbon detection card and reading a result;

preferably, a reverse transcription system in step (1) is 20 P L; wherein a volume ratio of the

reaction liquid to the RNA is 3: 1, and a reaction condition is as follows: warmly bathing in a

metal bath at a constant temperature of 38C for 10-15 min; and

preferably, the buffer solution in step (2) is 1% PBS-T.
10. An application of the detection method of any one of claims 8-9 in detecting Hepatitis E

Virus and Hepatitis A Virus.

Fig. 1 Drawings

Fig. 2 Drawings

Fig. 3 Drawings

Fig. 4 Drawings