AU2021103861A4

AU2021103861A4 - Method and kit for differentially detecting porcine pseudorabies vaccine virus and wild virus

Info

Publication number: AU2021103861A4
Application number: AU2021103861A
Authority: AU
Inventors: Wenlong Duan; Shenyang Gao; Dandan Li; Hui Li; Zongshuai Ling; Jiaying Wang; Shude Wang; Suijia Wang; Zhenzhu Weng; Enhui Zha; Tiezhong Zhou
Original assignee: Jinzhou Medical University
Current assignee: Jinzhou Medical University
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-08-26
Anticipated expiration: 2029-07-05

Abstract

Disclosed is a kit for detecting a UL52 gene and/or a gE gene of a porcine pseudorabies virus, including: a forward primer, a middle probe and a reverse primer which are used to amplify a UL52 gene sequence of the porcine pseudorabies virus and/or a forward primer, a middle probe and a reverse primer which are used to amplify a gE gene sequence of the porcine pseudorabies virus by means of recombinase polymerase amplification, the conventional reagents for recombinase polymerase amplification, and a colloidal gold test strip which includes a sampling pad, a control line, a No. 1 detection line and/or No. 2 detection line. Through the cooperation of the control line, the detection lines, and the primer and probe labeling, a UL52 gene and/or a gE gene of a porcine pseudorabies virus in a sample can be detected rapidly, sensitively and specifically by using the kit. - 37- NFO excises THF, and DNA polymerase Principle ofNFO-RPA extends from the excision site and amplifies a template QNFO (Endonuclease V) FAM Forwardprimer 5 Nucleic acid probe y THF VBlockIC3-spacer 5 - 3 5 labeled with FAM A. 3' 5' Perform isothermal amplification on the template at 38C for 20 Reverse primer min to obtain amplified products labeled with Biotin or Dig FAM FAM Amplified product of a UL52 gene of PRV Amplified product of a gE gene of PRV 8.otrn Principle of colloidal-gold test strip detection Colloidal-gold labeling particles Anti-FAM EFAM jAnti-FAM AM Amplified product of Amplified product of agE gene of PRV aUL52 gene of PRV Colloidal-gold labeling particles H Anti FAM Dig V n Anti. Anti FAM Y Anti-Dig Boin-Ligand Control line of test strip detection No. 2 detection line No.1 detection line * FAMlabeling 'K Coating anti-antibody(Anti-Anti-FAM) * Biotin labeling +( Coating digoxin antibody(Anti-Dig) FAM antibody labeled *Dig labeling ( Coating biotin binding ligand(Biotin-Ligand) by colloidal-gold Fig. 2 - 43-

Description

NFO excises THF, and DNA polymerase Principle ofNFO-RPA extends from the excision site and amplifies a template QNFO (Endonuclease V) FAM Forwardprimer 5 Nucleic acid probe y A. THF VBlockIC3-spacer 5- 3 5 labeled with FAM

3' 5' Perform isothermal amplification on the template at 38C for 20 Reverse primer min to obtain amplified products labeled with Biotin or Dig FAM FAM Amplified product of a UL52 gene of PRV Amplified product of a gE gene of PRV

8.otrn

Principle of colloidal-gold test strip detection Colloidal-gold labeling particles Anti-FAM jAnti-FAM EFAM AM

Amplified product of Amplified product of agE gene of PRV aUL52 gene of PRV

Colloidal-gold labeling particles H Anti FAM

Anti. Anti FAM Y Dig

Anti-Dig V n Boin-Ligand

Control line of test strip detection No. 2 detection line No.1 detection line

* FAMlabeling 'K Coating anti-antibody(Anti-Anti-FAM) * Biotin labeling +( Coating digoxin antibody(Anti-Dig) FAM antibody labeled *Dig labeling ( Coating biotin binding ligand(Biotin-Ligand) by colloidal-gold

Fig. 2

METHOD AND KIT FOR DIFFERENTIALLY DETECTING PORCINE PSEUDORABIES VACCINE VIRUS AND WILD VIRUS TECHNICAL FIELD

The present disclosure belongs to the technical field of biological nucleic acid

molecular detection, and relates to a method and kit for differentially detecting a

porcine pseudorabies vaccine virus and wild virus by combining recombinase

polymerase amplification (RPA) with colloidal-gold immunochromatography assay

(GICA). BACKGROUND

Animal inspection and quarantine has become a hot issue concerned by the

international community in recent years. China is a major country in animal

husbandry, and ranks first in the world in terms of pig breeding and consumption.

Viral diseases that cause sow abortion bring huge economic losses to the pig breeding

industry every year. Currently, there are several methods for detecting porcine viral

infection diseases: conventional polymerase chain reaction (PCR), fluorescent

quantitative PCR, a virus isolated culture and identification method, electron

microscopy observation, enzyme linked immunosorbent assay, immunofluorescence

assay, etc. In clinical practice, most of the diseased pigs are infected by mixed viruses.

The above methods are expensive and inconvenient to operate, which are not

conducive to on-site rapid detection of multiple batches at the same time.

Recombinase polymerase amplification (RPA) developed by TwistDx Inc, UK, is

a novel rapid isothermal nucleic acid molecule amplification technology and known

as a nucleic acid detection technology that can replace PCR. The technology mainly

includes the following steps: recombinases can tightly bind to DNA primers at a

constant temperature of 37C to form polymers of the enzymes and the primers, and

the primers melt template DNA with the help of single-stranded DNA binding (SSB)

after identifying completely complementary sequences on the template DNA, and

form new DNA complementary strands under the action of a DNA polymerase.

Reaction products also increase exponentially, and an amplified fragment that can be

detected by agarose gel electrophoresis is usually obtained within 1 hour. The whole

reaction is simple and rapid, and because it does not require high temperature cycles,

it is especially suitable for use in non-laboratory detection sites with a large number of

samples.

Molecular detection methods of a porcine pseudorabies virus include PCR,

fluorescent quantitative PCR, and LAMP, and immunologic detection methods of a

porcine pseudorabies virus includes ELISA and colloidal-gold

immunochromatography assay. Among the existing isothermal nucleic acid in-vitro

amplification methods, loop-mediated isothermal amplification (LAMP) is a

relatively mature novel technology. The core of LAMP is the use of a strand

displacement DNA polymerase. When double strands of DNA are in dynamic

equilibrium at 65°C, primers in the reaction can produce complementary sequences

on the same strand through a strand replacement reaction similar to rolling circle

replication round and round to form amplified products with different sizes. LAMP is

characterized in that 4 to 6 primers are needed, and a DNA amplification reaction is

performed at a constant temperature of 65°C under the action of a protein. The

reaction sensitivity of LAMP is higher than that of nested PCR, and the specificity is

high. If a fluorescent dye is used, the amplified products of the reaction can further be

observed directly with naked eyes.

Defect 1: the primers used by LAMP are relatively complicated, and special

software is required to design 4 to 6 primers, so the difficulty of the design is high and

the defect of excessive primer dimers of the amplified products is easily caused.

Defect 2: LAMP is prone to non-specific amplification. Due to the relatively

short length and relatively large number of primers in the same reaction, the mismatch

rate of the primers is easily increased, thereby causing the defect of false positive

reactions.

Defect 3: the amplified products obtained by LAMP are fragments with different

sizes which cannot be cloned and sequenced directly, and can only be used to

determine whether a target gene exists, which is also the biggest limitation of LAMP.

Defect 4: because of the large number of primers, LAMP can only perform

amplification reaction, and cannot perform double-amplified nucleic acid differential

diagnosis of different genotypes of the same pathogen, such as the distinguish

between the porcine pseudorabies wild virus and vaccine virus.

Corresponding terms: porcine pseudorabies virus (PRV), Recombinase

polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP),

and colloidal-gold immunochromatography assay (GICA).

SUMMARY

The present disclosure is directed to provide a practical detection method capable

of rapidly and differentially detecting nucleic acid of a porcine pseudorabies virus on

site. Primers and probes used in the nucleic acid qualitative detection method are both

for characteristic fragment areas of the porcine pseudorabies virus (PRV). The present

disclosure can differentially diagnose the porcine pseudorabies vaccine virus and wild

virus, and the specificity is very high.

The present disclosure is implemented according to the following technical

solution: an on-site rapid detection method for differentially diagnosing a porcine

pseudorabies virus by combining RPA and GICA, and its main solutions are as

follows: the nucleic acid on-site rapid differential diagnose technology includes

primers and probes for characteristic fragments of the porcine pseudorabies virus, and

the primers and the probes are selected from the characteristic fragments of the

porcine pseudorabies virus (PRV). The primers and the probes are designed according

to characteristics of PRV, are synthesized by means of artificial chemistry, and are

added with DNA fingerprint sequences with specific sizes and molecular labeling.

More specifically, the present disclosure provides a kit for detecting a UL52 gene

and/or a gE gene of a porcine pseudorabies virus, including: reagents required by

recombinase polymerase amplification for amplifying a UL52 gene sequence and/or a

gE gene sequence of the porcine pseudorabies virus, a reagent required by a

colloidal-gold test strip for showing an amplified product of the UL52 gene and/or an amplified product of the gE gene of the porcine pseudorabies virus, and the colloidal-gold test strip.

In some implementations, the reagents required by recombinase polymerase

amplification for amplifying the UL52 gene sequence of the porcine pseudorabies

virus include:

a first direction primer UL52primerl for the UL52 gene of the porcine

pseudorabies virus;

a middle probe UL52probe for the UL52 gene of the porcine pseudorabies virus,

the UL52probe being connected with a first labeled molecule forward and a protection

label for preventing polymerization reversely, and including an artificial base

analogue which can be identified and excised by an enzyme with DNA damage repair

activity in the middle;

a second direction primer UL52primer2 for the UL52 gene of the porcine

pseudorabies virus, the UL52primer2 being connected with a second labeled molecule

forward,

wherein the UL52 primer and the UL52primer2 are for the same direction of

the UL52 gene, and the UL52probe for the UL52 gene is located between the

UL52primerl and the UL52primer2; and/or the reagents required by recombinase

polymerase amplification for amplifying the gE gene sequence of the porcine

pseudorabies virus include:

a first direction primer gEprimerl for the gE gene of the porcine pseudorabies

virus;

a middle probe gEprobe for the gE gene of the porcine pseudorabies virus, the

gEprobe being connected with the first labeled molecule forward and the protection

label for preventing polymerization reversely, and including the artificial base

analogue which can be identified and excised by the enzyme with DNA damage repair

activity in the middle;

a second direction primer gEprimer2 for gE gene of the porcine pseudorabies

virus, the gEprimer2 being connected with a third labeled molecule forward, wherein the gEprimerl and the gEprobe are for the same direction of the gE gene, and the gEprobe for the gE gene is located between the gEprimerl and the gEprimer2; and the second labeled molecule and the third labeled molecule are labeled molecules with different binding characteristics.

In some implementations, the colloidal-gold test strip includes a sampling pad, a

control line, a No. 1 detection line and/or No. 2 detection line;

the sampling pad includes a first labeled molecule specific conjugate labeled by

colloidal-gold particles;

the control line is coated with a specific conjugate of the first label molecule

specific conjugate;

the No. 1 detection line is coated with a second labeled molecule specific

conjugate; and

the No. 2 detection line is coated with a third labeled molecule specific

conjugate.

In some implementations, the first labeled molecule is a fluorescein molecule

(FAM), and the first labeled molecule specific conjugate is an FAM antibody;

the second labeled molecule is biotin, and the second labeled molecule specific

conjugate is streptavidin;

the third labeled molecule is digoxin, and the third labeled molecule specific

conjugate is a digoxin antibody;

the specific conjugate of the first labeled molecule specific conjugate is an

antibody of the FAM antibody;

the protection label for preventing polymerization is C3-spacer; and

the artificial base analogue which can be identified and excised by the enzyme

with DNA damage repair activity is tetrahydrofuran.

In some implementations, a sequence of the UL52primerl is as follows:

5'-GCGCGCCATGGAGTACTTTTACACGTCCCAGTGCCC-3'; a sequence of the UL52primer2 is as follows:

5'-[Biotin]-ACCTCGTCAACGTCAACCTGGGCAGCCTCTCGGAGCT-3'; a sequence of the UL52probe is as follows:

5'-[FAM]-GCGCTCAGCGAGGCCGAGCTCGAGTTCTACCGCTT-[THF]-CT

CTTCGCCTTCCTCT-[C3-spacer]-3';

a sequence of the gEprimerl is as follows:

5'-GACGACGCCCGGGCGGCTGTTTGTGCTGGCGCTGGG-3'; a sequence of the gEprobe is as follows:

5'-[FAM]-TGCTCCCGGCGCCGGGCGGCCTCGCGGCCGTTCC-[THF]-GG TGCCGACGCGGGCGC-[C3-spacer]-3'; a sequence of the gEprimer2 is as follows:

5'-[Dig]-CGTCGTAGTAGTCCTCGTGCGTGGGCAGGCTGGTGT-3', wherein Biotin represents a biotin molecule, Dig represents a digoxin molecule,

FAM represents the fluorescein molecule (FAM), and THF represents a

tetrahydrofuranmolecule.

In some implementations, the kit further includes an NFO enzyme, a

recombinase uvsX, an accessory protein uvsY, a Gp 32 protein; a Bsu DNA

polymerase, dNTPs, dithiothreitol, adenosine triphosphate, trimethylglycine

trimethylglycocoll, polyethylene glycol 20000, creatine kinase, phosphokinase,

potassium acetate and magnesium acetate.

The present disclosure further provides a method for detecting a UL52 gene

and/or a gE gene of a porcine pseudorabies virus, and the method is used for a

non-diagnostic purpose, including: amplifying a UL52 gene sequence and/or a gE

gene sequence of the porcine pseudorabies virus by means of recombinase

polymerase amplification, and showing an amplified product of the UL52 gene and/or

an amplified product of the gE gene of the porcine pseudorabies virus by using a

colloidal-gold test strip.

In some implementations, the method includes the following steps:

(1) extracting nucleic acid of a biological material which may contain a UL52

gene and/or a gE gene of a porcine pseudorabies virus;

(2) performing recombinase polymerase amplification on the nucleic acid

obtained at step (1) by using the UL52primerl, the UL52probe and the UL52primer2,

and/or the gEprimerl, the gEprobe and the gEprimer2 of any one of claims 2 to 6; and

(3) showing an amplified product obtained at step (2) by using the colloidal-gold

test strip of any one of claims I to 6.

In some implementations, step (2) includes the following substeps:

(i) taking 2 L of the extracted nucleic acid sample obtained at step (1) and

adding the same into a 0.2 mL centrifuge tube, and then adding 45.5 L of an RPA

reaction liquid, 100 ng/L recombinase uvsX, 50 ng/L accessory protein uvsY, 100

ng/4L Gp 32 protein, 25 ng/L Bsu DNA polymerase, 0.5 mM dNTPs, 1 mM

dithiothreitol, 3 mM adenosine triphosphate, 100 mM trimethylglycine

trimethylglycocoll with a pH of 8.0, 5% polyethylene glycol 20000, 100 ng/L

creatine kinase, 20 mM phosphokinase, and 80 mM potassium acetate into the

centrifuge tube,

wherein the RPA reaction liquid contains labeled primers and labeled probes for

specific nucleic acid amplification and detection, and specifically contains the

following components: 50 pM UL52primerl, 50 pM UL52primer2, 10 pM

UL52probe, 50 pM gEprimerl, 50 pM gEprimer2, 10 pM gEprobe, and 10 units of

NFP enzymes; and

(ii) uniformly mixing the above reaction liquid, adding 2.5 L of 100 mmol/L

magnesium acetate, uniformly mixing the mixture and placing the same in a

thermostatic metal bath for warm bathing at 38C for 15 to 30 min to obtain an

amplified product which is used as an amplified sample to be detected.

In some implementations, step (3) includes the following substeps:

(a) taking a colloidal-gold test strip and balancing the same at room temperature;

(b) adding a sample: taking the amplified product obtained at step (2) and

diluting the same with pure water, and dropwise adding the diluted amplified product

to a sample hole of the colloidal-gold test strip; and

(c) reading a result.

Solved problem 1: improvement of the specificity of the isothermal nucleic acid

amplification reaction. RPA reaction only uses a pair of primers which are longer by

about ten bases than those used in conventional PCR and LAMP, and it can be known

from the principle of base complementary that the longer the primer, the higher the

specificity, and therefore RPA greatly improves the specificity and reliability of the

nucleic acid amplification reaction. Two sets of primers and specifically labeled

probes for identification are designed by respectively taking the UL52 gene and the

gE gene of the porcine pseudorabies virus as target genes to realize amplification of

the UL52 gene and the gE gene at the same time in the same reaction.

Solved problem 2: realization of on-site rapid differential detection of PRV.

Results of the RPA reaction are combined with the GICA test strip, thereby further

improving the conveniences of detection and visualizing the amplification detection

results. The detection results of the amplified UL52 and gE gene fragments can be

directly distinguished, and corresponding detection lines are designed by respectively

taking the UL52 gene as an internal reference typical vaccine virus and/or the gE gene

as a wild virus to realize the differential detection of a porcine pseudorabies virus.

Solved problem 3: realization of on-site rapid molecular detection of a porcine

pseudorabies virus. RPA is a method for amplifying nucleic acid at a constant

temperature, and does not require a PCR instrument, thereby solving the defect of

high cost of PCR. The RPA reaction can finish within about 20 min, which saves a lot

of time compared with PCR. The combination of RPA and a GICA test strip makes

test results directly readable on site without relying on an expensive gel imaging

system and a device such as a computer, thereby realizing on-site repaid molecular

detection of the porcine pseudorabies virus.

Compared with the prior art, the present disclosure has the following beneficial

effects:

1. The present disclosure can differentially detect the porcine pseudorabies

vaccine virus and wild virus at the same time.

2. The present disclosure uses specific long primers and probes to amplify

nucleic acid of a porcine pseudorabies virus so as to obtain an amplified product,

which improves the specificity and reliability of a detection result.

3. The present disclosure omits the process of judging a result of an amplified

product after gel electrophoresis in original PCR or LAMP, and combines RPA with a

GICA test strip, which makes a detection result directly readable on site, thereby

realizing on-site rapid molecular detection of the porcine pseudorabies virus.

4. The present disclosure realizes on-site rapid molecular detection of a porcine

pseudorabies virus, and RPA is a method for amplifying nucleic acid at a constant

high cost of PCR.

5. Because RPA reaction can finish within about 20 min, the present disclosure

saves a lot of time compared with PCR.

Beneficial effect 1: RPA can be used to perform single-molecule nucleic acid

detection at room temperature within 15 to 20 min. The technology does not require

high hardware equipment, and is particularly suitable for on-site rapid detection and

diagnosis in the fields of food safety, veterinary medicine, biological defense,

agriculture, etc. Generally, the conventional PCR must involves three steps of

degeneration, annealing and extension to realize nucleic acid amplification, and the

PCR instrument essentially is a device for controlling temperature rise and fall. The

optimum temperature for RPA reaction is between 37°C and 42°C, and the RPA

reaction can be performed at room temperature without degeneration. Therefore, the

amplification speed can be greatly accelerated, and thus rapid detection is possible.

Beneficial effect 2: Because RPA does not require a temperature control device,

RPA can truly realize portable and rapid nucleic acid detection. The sensitivity of RPA

detection is very high, and RPA can amplify a template with trace amount of nucleic

acid (especially DNA) to a detectable level and obtain about 1012 amplified products

from a single template molecule. RPA does not require complicated sample treatment,

and thus it is suitable for detection in a site where nucleic acid cannot be extracted.

Beneficial effect 3: RPA can amplify both DNA and RNA without an additional

cDNA synthesis step. Through RPA, not only end-point detection of the amplified

product can be performed, but also results can be directly read after a detection signal

of the amplified product is amplified by a colloidal-gold immunochromatographic test

strip.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of RPA.

Fig. 2 is a schematic diagram of reading an NFO-RPA amplified product on a

GICA test strip.

NFO-RPA is an improved method of adding an NFO enzyme (endonuclease IV)

and a specific molecule labeled nucleic acid probe as well as a reverse amplification

primer with modified ends on the basis of an RPA system. As shown in Fig. 2, a

design principle of the molecule labeled nucleic acid probe is to label a 5' end of the

nucleic acid probe with FAM (fluorescein molecule), introduce a heterologous

nucleotide residue THF (tetrahydrofuran) in the middle, and add a blocker C3-spacer

to a 3' end of the nucleic acid probe to prevent a Bsu polymerase from being induced

to amplify non-target DNA. Only when the probe and the DNA strand are in

complementary binding, the NFO enzyme with DNA damage repair activity identified

and excises THF at a specific site, the Bsu polymerase continues to perform

amplification and extend from the site, and a specific amplicon product with a

forward 5' end labeled with the FAM probe and a reverse 5' end labeled with Biotin

(biotin) or Dig (digoxigen) is finally formed.

A detection region of the GICA test strip includes a No. 1 detection line

corresponding to a position coated with a Bitotin specific conjugate (Biotin-Ligand), a

No. 2 detection line corresponding to a position coated with an Dig antibody, and a

control C line corresponding to a position coated with an antibody of an FAM

antibody (Anti-Anti-FAM), respectively.

After the NFO-RPA amplicon product is dropwise added to a sampling hole, the

FAM antibody labeled by colloidal-gold particles on a sampling pad flows with the liquid sample and at the same time specifically binds to a 5' FAM molecule of the

NFO-RPA amplicon. Meanwhile, the other end of the amplicon is specifically

captured by the molecule coated on the No. 1 line or No. 2 line, respectively, to form

a sandwich-typed compound, the FAM antibody labeled by colloidal-gold particles

which does not specifically bind to the amplicon binds to the antibody of the FAM

antibody coated on the control C line to form a control detection line.

Fig. 3 is a diagram of a reading and judgment method of an NFO-RPA-GICA

combined test strip.

After the NFO-RPA amplicon product is dropwise added to the sampling hole, a

detection result is judged within 10 min, and a judgment method is as follows: (1)

when the control C line appears, it indicates that the test is effective, and at the

moment, when the No. 1 and No. 2 detection lines appear simultaneously, it indicates

that a PRV wild virus is detected, and thus a detection result is positive; (2) when the

control C line appears, it indicates that the test is effective, and at the moment, when

only No. 1 detection line appears, it indicates that a PRV vaccine virus is detected,

and thus a detection result is positive; (3) when the control C line appears, it indicates

that the test is effective, and at the moment, when neither of the No. 1 and No. 2

detection lines appears, it indicates that neither of the PRV vaccine virus and wild

virus is detected, and thus a detection result is negative; and (4) when the control C

line does not appear, it indicates that the test is ineffective, and thus a detection result

is meaningless.

Fig. 4 is a diagram of results of an experiment on the sensitivity of

NFO-RPA-GICA detection.

From top to bottom, a first test strip is a blank control which is a GICA detection

result of an NFO-RPA amplified product of an empty plasmid pMD18-T used as a

template; and second to sixth test strips are respectively GICA detection results of

pMD18-T-UL52 and pMD18-T-gE plasmid templates which respectively contain a

PRV UL52 gene and a gE gene and are diluted according to five different

concentrations of 3.6x10, 3.6x102, 3.6x10, 3.6x104, 3.6x105 copy/pl after

RPA-GICA amplification.

Fig. 5 is a diagram of results of a comparative experiment on the sensitivity of

PCR detection of the PRV UL52 gene.

In the figure, the gel density is 1.5%, a No. 1 lane represents a blank control

template which is pMD18-T plasmid at 1x105 copy/pl; and No. 2 to No. 6 lanes

respectively represent detection results of the sensitivity of PCR amplification of

pMD18-T-UL52 plasmid templates at concentrations of 3.6x10, 3.6x102, 3.6x103,

3.6x104, 3.6x105 copy/pl. M represents a DNA marker DM 2000.

Fig. 6 is a diagram of results of a comparative experiment on the sensitivity of

PCR detection of the PRV gE gene.

In the figure, the gel density is 1.5%, a No. 1 lane represents a blank control

template which is a pMD18-T plasmid at 1x105 copy/pl; and No. 2 to No. 6 lanes

pMD18-T-gE plasmid templates at concentrations of 3.6x10, 3.6x102, 3.6x103,

3.6x104, 3.6x105 copy/pl. M represents a DNA marker DM 2000.

Fig. 7 is a diagram of results of an experiment on the specificity of

NFO-RPA-GICA detection.

From top to bottom, a first test strip is a nucleic acid detection result of a PRV

wild virus; a second test strip is a nucleic acid detection result of a PRV vaccine virus;

a third strip is a nucleic acid detection result of a classical swine fever virus (CSFV); a

fourth strip is a nucleic acid detection result of a porcine reproductive and respiratory

syndrome virus (PRRSV), a fifth test strip is a nucleic acid detection result of a

porcine circovirus type 2 (PCV2); a sixth test strip is a nucleic acid detection result of

a porcine parvovirus (PPV); and a seventh test strip is a nucleic acid detection result

of a porcine Japanese encephalitis virus (PJEV).

Fig. 8 is a diagram of results of a comparative experiment on the specificity of

PCR detection of a UL52 gene of PRV.

In the figure, the gel density is 1.5%, M represents a DNA maker DM2000; a No.

1 lane is a PCR detection result of the UL52 gene of the PRV vaccine virus, and No. 2

to No. 6 lanes are nucleic acid PCR controls of CSFV, PRRSV, PCV, PPV and PJEV,

respectively.

Fig. 9 is a diagram of results of a comparative experiment on the specificity of

PCR detection of a gE gene of PRV.

In the figure, the gel density is 1.5%, M represents a DNA marker DM2000; a

No. 1 lane is a PCR detection result of the gE gene of the PRV wild virus , and No. 2

to No. 6 lanes are nucleic acid PCR controls of CSFV, PRRSV, PCV, PPV and PJEV,

respectively.

DETAILED DESCRIPTION

To better explain the technical solutions of the present disclosure, embodiments

of the present disclosure will be described below in details with reference to the

accompanying drawings. The following embodiments are used to further explain the

present disclosure, but should not be understood as a fixation or limitation to the

present disclosure. Unless otherwise defined, technical features used in the

embodiments may be replaced with other known technical features in the art that have

equivalent or similar functions or effects without departing from the concept of the

present disclosure.

1. Inventive concept

1.1 Overview of a UL52 gene of a porcine pseudorabies virus

Pseudorabies (PR) is a highly contagious infectious disease caused by a

pseudorabies virus (PRV). PRV is a member of Varicellovirus of Alphaherpesvirinae

of Herpesviridae. A virus particle is round with a diameter of 150 to 180 nm. A virus

structure is composed of four parts, i.e. a central core marrow, a capsid, a

mesomesenchyme and an envelope. A genome of PRV is a linear double-stranded

DNA molecule with a size of about 150 kb. The genome of PRV includes a unique

long sequence (UL) and a short sequence (US), and there are a terminal repeated

sequence (TRR) and an internal repeated sequence (IRS) at two sides of US. PRV

genes are named according to regions where they are located and the order in which

they are found, and can also be named according to proteins encoded by them.

Non-structural protein genes located in the UL region include UL9 that can bind to a

replication origin, UL30 that encodes a DNA polymerase, UL 52 that encodes a helicase, UL54 that participates in transcription regulation, etc. It can be found through Blast sequence comparison analysis that some regions of the UL 52 that encodes the helicase are relatively extremely conservative, and are more conservative than other encoding genes required by replication, such as reported PRV virulence-related factor encoding genes gB and gD, and thus the UL52 gene is selected as a target gene for PRV specific diagnosis. Currently, there is no report on the use of the UL52 gene as the target gene for PRV diagnosis.

1.2 Overview of a gE gene of the pseudorabies virus

The genome of PRV encodes a total of 16 membrane proteins among which 11

membrane proteins are modified by 0-linked or N-linked sugars and are named gB,

gC, gD, gE, gG, gH, gI, gK, gL, gM, and gN, respectively, and 4 proteins are not

glycosylated (UL20, UL43, US9, UL24 and UL34). The gE and the gI are not

necessary for virus proliferation, but they are main virulence genes of PRV. If the gE

is deleted, although PRV cannot invade the animal's tertiary neurons, but PRV still

can invade host cells and the immunogenicity of the virus is not effected. Therefore,

by taking advantage of the deletion characteristic of the gE gene of PRV, numerous gE

gene-deleted vaccines have been developed and widely used in the current breeding

industry. Currently, in China, the commercially available and most widely used

vaccine is a PRV attenuated freeze-dried vaccine (Bathar-k61 strain) which is a dual

gE/gI gene-deleted attenuated vaccine, and its gene deletion blocks the possibility of

virulence recovery of the attenuated strain, so that the safety of the vaccine is

improved to a large extent. The attenuated live vaccine is originally obtained by

isolation of a wild strain, repeated passage of non-pig-derived cells, addition of a

mutagen or adaptation to chicken embryos, and repeated passage of cells at a

relatively high culturing temperature. In China and other countries, a variety of gE

gene-deleted vaccines have been developed, which provide a powerful tool for the

purification of pseudorabies. The combination of the use of the gE gene-deleted

vaccines in pig farms and necessary molecular biological monitoring methods

provides an important scientific basis for the prevention and control of the disease.

1.3 Significance of differential diagnosis

Based on the above, it can be seen that the integrated detection of a UL52 gene and a gE gene of a pseudorabies virus in a sample at the same time has important clinical diagnostic significance for the prevention and control of pseudorabies. According to the actual situation of the use of the pseudorabies vaccines in a pig farm, an accurate and rapid judgment can be made on whether infection of a pseudorabies wild virus in the pig farm exists. That is, in a first case, in the absence of the pseudorabies gE gene-deleted vaccines, the presence of infection of the pseudorabies wild virus in a pig farm can be basically determined by detecting the two kinds or at least one kind of genes by using the present method. In a second case, in the case of using the pseudorabies gE gene-deleted vaccines, if a pig is detected by using the present method and a detection result shows that the gE gene is negative and the UL52 gene is positive, it only proves that the pig is not infected with the pseudorabies wild virus, and the case of positive gE gene and negative UL52 gene hardly occurs; if the detection result shows that the two genes are both negative, it may indicate vaccination failure or elimination of virus particles after protection immunity is produced; and if the detection results shows that the two genes are both positive, it indicates that the pig is infected with the pseudorabies wild virus. 1.4. Core concepts RPA is rapid, accurate, economical, portable, etc. The details are as follows: RPA can be used to perform single-molecule nucleic acid detection at room temperature within 15 to 20 min. The technology has low requirements on hardware equipment, and is particularly suitable for on-site rapid detection and diagnosis in the fields of food safety, veterinary medicine, biological defense, agriculture, etc. Generally, the conventional PCR must involves three steps of degeneration, annealing and extension to realize nucleic acid amplification, and the PCR instrument essentially is a device for controlling temperature rise and fall. The optimum temperature for RPA reaction is between 37°C and 42°C, and the RPA reaction can be performed at room temperature without degeneration. The PCR speed can be greatly accelerated, thereby meeting the requirements of the on-site rapid detection technology for convenience.

Because RPA does not require a temperature control device, RPA can truly

realize portable and rapid nucleic acid detection. The sensitivity of RPA detection is

very high, and RPA can amplify a template with trace amount of nucleic acid

(especially DNA) to a detectable level and obtain about 1012 amplified products from

a single template molecule. RPA does not require complicated sample treatment, and

thus it is suitable for detection in a site where nucleic acid cannot be extracted.

RPA can amplify both DNA and RNA without an additional cDNA synthesis step.

Through RPA, not only end-point detection of the amplified product can be performed,

but also results can be directly read after a detection signal of the amplified product is

further amplified by a colloidal-gold immunochromatographic test strip.

The principle of colloidal-gold immunochromatography assay (GICA) is as

follows: a specific antigen or antibody is fixed on a film in a form of strip, a

colloidal-gold labeling reagent (antibody or monoclonal antibody) is absorbed on a

binding pad, a sample to be detected moves forward through capillarity after being

added to a sample pad at an end of a test strip, dissolves and reacts with the

colloidal-gold labeling reagent on the binding pad, when moving to a fixed antigen or

antibody region, a conjugate of the sample to be detected and the colloidal-gold

labeling reagent specifically binds to the antigen or antibody and is trapped, and

aggregates on a detection zone. A color developing result can be observed by naked

eyes. The method has been applied to a diagnostic test strip, which is very convenient

to use. The reliability of the method depends on whether the sensitivity and specificity

of the used raw materials such as a monoclonal antibody, a polyclonal antibody, an

antigen, a hapten, a protein chimera, and a colloidal-gold can meet the highest

standards, which is the most important standard for the quality of the colloidal-gold

labeling reagent.

The convenience is as follows:

1. After a test strip is taken out, a sample to be detected is dropwise added to a

sample slot, and a detection result will be shown within 10 min and can be judged

directly by naked eyes.

2. A shelf life of a test strip in a vacuum packaging bag is more than half a year.

It is ready to use, which is very convenient to use and carry.

3. Test strips can be mass-produced, which facilitates the formulation of

detection standardization.

4. Mass-produced test strips can greatly reduce economic costs, and thus on-site

detection can be an acceptable routine diagnostic item.

Based on the above concepts, the present disclosure provides an on-site rapid

detection method for differentially diagnosing a porcine pseudorabies virus by using

RPA, including the following core steps:

A. nucleic acid of a blood or nasal secretion liquid sample of a diseased pig or

dead pig suspected to have been infected with the above virus is extracted to obtain a

sample template to be detected;

B. the sample template to be detected is mixed with a RPA reaction liquid at a

constant temperature of 38C for 20 min to obtain an amplified product; and

C. detection lines of amplified products of the UL52 gene and gE gene of the

porcine pseudorabies virus are respectively arranged on a colloidal-gold test strip, the

amplified product is diluted and dropwise added to the colloidal-gold test strip, a

detection result is read within 10 min to judge an infection status of the pig.

Technical key point 1: molecule labeled nucleic acid probes and primers that can

specifically identify the UL52 gene and the gE gene of the porcine pseudorabies virus

are designed.

Technical key point 2: reaction conditions are optimized, the molecule labeled

nucleic acid probes and primers of the UL52 gene and the gE gene are mixed with an

RPA reagent to prepare a MasterMix reaction liquid, thereby realizing convenient

operation, reducing an operating step of sampling, and reducing the reaction pollution

probability.

Technical key point 3: a colloidal-gold (GICA) test strip is designed to be coated

with labeled molecules that can specifically capture the amplified products of the

UL52 gene and the gE gene, thereby realizing the differential detection of the porcine

pseudorabies vaccine virus and wild virus.

2. Design of primers

To achieve the above objectives, the present disclosure designs primers and

probes for the UL52 gene and gE gene of the porcine pseudorabies virus. Through

comparative analysis of homologous sequences of the UL52 gene and the gE gene of

JF797217.1, JF797219.1, JQ809328.1, KJ789182.1, KP098534.1, KM189914.3,

KP722022.1, KM189912.1, KX423960.1, KT824771.1, and KC981239.1 from

GenBank, a conservative core region is determined, primers and probes are designed

for the region, and the region is used as an universal gene detection target for

detecting a porcine pseudorabies virus. All of the primers and probes are synthesized

by Shanghai Bioengineering Co., Ltd.

First, primers and probes of an on-site rapid detection method for differentially

diagnosing a porcine pseudorabies virus by using RPA are provided, which include

the following sequence sets:

1) the following two sets of sequences of forward and reverse primers and

molecule labeled nucleic acid probes are included:

a forward primer, a reverse primer and a molecule labeled nucleic acid probe for

the UL52 gene of the porcine pseudorabies virus are as follows, respectively:

UL52primerl (a sequence is shown as SEQ ID NO. 1)

5'-GCGCGCCATGGAGTACTTTTACACGTCCCAGTGCCC-3' UL52primer2 (a sequence is shown as SEQ ID NO. 2)

5'-[Biotin]-ACCTCGTCAACGTCAACCTGGGCAGCCTCTCGGAGCT-3'; UL52probe (a sequence between FAM and THF is shown as SEQ ID NO. 3; and

a sequence between THF and C3-spacer is shown as SEQ ID NO. 4)

5'-[FAM]-GCGCTCAGCGAGGCCGAGCTCGAGTTCTACCGCTT-[THF]-CT

CTTCGCCTTCCTCT-[C3-spacer]-3'

and

the gE gene of the porcine pseudorabies virus are as follows, respectively:

gEprimerl (a sequence is shown as SEQ ID NO. 5)

5'-GACGACGCCCGGGCGGCTGTTTGTGCTGGCGCTGGG-3' gEprimer2 (a sequence is shown as SEQ ID NO. 6)

5'-[Dig]-CGTCGTAGTAGTCCTCGTGCGTGGGCAGGCTGGTGT-3'; gEprobe (a sequence between FAM and THF is shown as SEQ ID NO. 7; and a

sequence between THF and C3-spacer is shown as SEQ ID NO. 8)

5'-[FAM]-TGCTCCCGGCGCCGGGCGGCCTCGCGGCCGTTCC-[THF]-GG TGCCGACGCGGGCGC-[C3-spacer]-3'; 3. Detecting steps

(1) Sample pretreatment

A sample may be a serum, a nasopharyngeal swab, a pharyngeal swab, or other

tissue of a pig; ( Serum sample: 5 ml of venous blood is collected and placed into a

mL screw plastic centrifuge tube with a washer (without an anticoagulant),

centrifuged under 1,000 g for 5 to 10 min, and subpackaged according to 100 L per

tube. @ Pharyngeal swab and nasopharyngeal swab specimens: pharyngeal swab

collection is to use a cotton swab to wipe the posterior pharyngeal walls of both sides

with a moderate force; and nasopharyngeal swab collection is to insert a cotton swab

parallel to the upper palate into the nostril to absorb secretions and swab the nostrils

of both sides. @ Tissue of a pathological material: a fresh pathological material with

a size of 3 to 5 mm3 is collected.

(2) Viral DNA extraction:

Viral DNA extraction was performed by using a rapid paramagnetic particle

extraction method which includes the following steps:

for a tissue sample, a tissue block with a size of 3 to 5 mm3 is taken by a scissor

and a tweezer and placed at a bottom of a 1.5 mL centrifuge tube, and fast ground by

a grinder to a muddy shape, 500 L of a lysis solution (10 mmol/L Tris.Cl (pH 8.0),

mmol/L EDTA, 150 mmol/L NaCl, 0.5% SDS, and 100 to 200 g/ml protease K)

is added into the tube, and the mixture is thoroughly oscillated and uniformly mixed;

for plasma, serum, ascites, cell culturing product and other liquid samples, 200 [

of the sample is directly taken and added into a 1.5 mL nuclease-free centrifuge tube,

500 pl of a lysis solution (10 mmol/L Tris.Cl (pH 8.0), 10 mmol/L EDTA, 150 mmol/L NaCL, 0.5% SDS, and 100 to 200 g/ml protease K) is added into the tube, and the mixture is thoroughly oscillated and uniformly mixed.

The sample mixture was lysed in a warm bath at 65 to 70°C for 5 min.

10 L of a thoroughly mixed magnetic bead solution (magnetic beads are labeled

with a layer of silicone plasmalemma, and a reaction principle is that under the

condition of high salt ion, the silicone plasmalemma binds to the nucleic acid by

means of positive and negative electric adsorption force, and in a low-salt ion

environment, the nucleic acid is eluted and separated to achieve the purpose of nucleic

acid separation) is added into the centrifuge tube, and the mixture is uniformly mixed

at room temperature for 10 min by inverting the centrifuge tube.

The tube is places on a magnetic stand for 1 min to allow the magnetic beads in

the tube to be absorbed, liquid in the tube is removed by using a pipettor, and the

centrifuge tube is removed.

500 pL of a protein-removed rinse solution (50 mM Tris-HCl with a pH of 8.0,

200 mM NaCl, and 50% ethanol solution) is added into the tube to allow the magnetic

beads to be resuspended, the centrifuge tube is placed on the magnetic stand for 1 min,

liquid in the tube is removed by using the pipettor, and the centrifuge tube is removed.

500 L of a washing solution (70% ethanol solution) is added into the tube to

allow the magnetic beads to be resuspended, the centrifuge tube is placed on the

magnetic stand for 1 min, liquid in the tube is removed by using the pipettor, and the

centrifuge tube is removed.

500 L of the washing solution (the 70% ethanol solution) is added again into the

tube to allow the magnetic beads to be resuspended, the centrifuge tube is placed on

the magnetic stand for 1 min, liquid in the tube is removed by using the pipettor, and

at the same time the centrifuge tube is kept on the magnetic stand so that the magnetic

beads continue to be absorbed and are dried in the air at room temperature for 5 min.

The centrifuge tube is removed from the magnetic stand, 50 to 100 L of eluent

(a TE buffer solution with a pH of 8.0, 10 mM Tris-HCl, and 1 mM EDTA) is added

into the tube to allow the magnetic beads to be resuspended, the tube is placed in a warm bath at 55C for 5 min, and is slightly shaken twice in the warm bath so that the nucleic acid (extracted virus genome DNA) is thoroughly eluted.

The centrifuge tube is placed on the magnetic stand for 1 min to allow the

magnetic beads to be are absorbed, and liquid is transferred into a new 1.5 ml RNA

enzyme-free centrifuge tube and used as an amplification template for later use.

(3) RPA reaction

Operating steps are as follows:

2 L of the amplification template obtained at step (2) is taken and added into a

0.2 mL centrifuge tube, 45.5 pL of an RPA reaction liquid (the reaction liquid contains

labeled primers and labeled probes for specific nucleic acid amplification and

detection, and specifically contains the following components: 50 pM UL52primerl,

pM UL52primer2, 10 pM UL52probe, 50 pM gEprimerl, 50 pM gEprimer2, 10

pM gEprobe, 10 units of NFO enzymes (endonuclease IV), 100 g/L recombinase

uvsX, 50 ng/L accessory protein uvsY, 100 ng/L Gp 32 protein (single-stranded

DNA binding (SSB)), 25 ng/4L Bsu DNA polymerase, 0.5 mM dNTPs, 1mM DTT

(dithiothreitol), 3 mM ATP (adenosine triphosphate), 100 mM Tricine

(trimethylglycine trimethylglycocoll) with a pH of 8.0, 0.5% PEG 20K (polyethylene

glycol 20000), 100 ng/L Creatine kinase (creatine kinase), 20 mM Phosphokinase

(phosphokinase), 80 mM Potassium acetate (potassium acetate)) is added into the tube,

the above reaction liquid is uniformly mixed and added with 2.5 L of an RPA

reaction primer fluid (containing 100 mmol/L magnesium acetate), and the mixture is

uniformly mixed and placed in a thermostatic metal bath for warm bathing at 38C

for 20 min to obtain an amplified product which is used as a sample to be detected.

The probe can continuously re-amplify a central small fragment by taking the

amplified product of the forward and reverse primers as a template. This design

greatly improves the specificity of the dual-labeled product, and also ensures the

specificity of subsequent colloidal-gold detection. In addition, to further improve the

anti-specificity of reaction, the concentration of the primers in the reaction system is

about five times higher than that of the labeled probe. Thus, there is enough template

amount in the final product for the probe to identify and bind, and the amount of the amplified product using the labeled probe and the reverse primer as a 5' end is sufficiently guaranteed, thereby providing a reliable basis for the sensitivity of subsequent colloidal-gold test strip detection. (4) Result detection and judgment A. Detection principle A principle of GICA detection is that as shown in Fig. 2, NFO-RPA is an improved method of adding a NFO enzyme (endonuclease IV) and a specific molecule labeled nucleic acid probe as well as a modified reverse amplification primer on the basis of an RPA system. As shown in Fig. 2, a design principle of the molecule labeled nucleic acid probe is to label a 5' end of the nucleic acid probe with FAM (fluorescein molecule), introduce a heterologous nucleotide residue THF (tetrahydrofuran) in the middle, and add a blocker C3-spacer to a 3' end of the nucleic acid probe to prevent a Bsu polymerase from being induced to amplify non-target DNA. Only when the probe and the DNA strand are in complementary binding, the NFO enzyme with DNA damage repair activity identifies and excises THF at a specific site, the Bsu polymerase continues to perform amplification and extend from the site, and finally a specific amplicon product with a forward 5' end labeled with the FAM probe and a reverse 5' end labeled with Biotin (biotin) or Dig (digoxigen) is formed. A detection region on the GICA test strip includes a No. 1 detection line corresponding to a position coated with a Bitotin specific conjugate (Biotin-Ligand), a No. 2 detection line corresponding to a position coated with an Dig antibody, and a control C line corresponding to a position coated with an antibody of an FAM antibody (Anti-Anti-FAM), respectively. After the NFO-RPA amplicon product is dropwise added to a sampling hole, the FAM antibody labeled by colloidal-gold particles on a sampling pad flows with the liquid sample and at the same time specifically binds to a 5' FAM molecule of the NFO-RPA amplicon. Meanwhile, the other end of the amplicon is specifically captured by the molecules coated on the No. 1 line or No. 2 line, respectively, to form a sandwich-typed compound, the FAM antibody labeled by colloidal-gold particles which does not specifically bind to the amplicon binds to the antibody of the FAM antibody coated on the control C line to form a control detection line.

Ajudgment method: as shown in Fig. 3, after the NFO-RPA amplicon product is

dropwise added to the sampling hole, a detection result is judged within 10 min, and

the judgment method is as follows: (1) when the control C line appears, it indicates

that the test is effective, and at the moment, when the No. 1 and No. 2 detection lines

appear simultaneously, it indicates that a PRV wild virus is detected, and thus a

detection result is positive; (2) when the control C line appears, it indicates that the

test is effective, and at the moment, when only No. 1 detection line appears, it

indicates that a PRV vaccine virus is detected, and thus a detection result is positive;

(3) when the control C line appears, it indicates that the test is effective, and at the

moment, when neither of the No. 1 and No. 2 detection lines appears, it indicates that

neither of the PRV vaccine virus and wild virus is detected, and thus a detection result

is negative; and (4) when the control C line does not appear, it indicates that the test is

ineffective and thus a detection result is meaningless.

B. Specific operation

Step 1: a colloidal-gold test strip is taken out, balanced at room temperature, and

marked with serial number;

Step 2, sample adding: 5 L of the sample to be detected at step (3) is taken,

diluted with 75 L of pure water, and dropwise added to a sample hole of the

colloidal-gold test strip.

Step 3, result reading: a detection result can be read after reaction is performed at

room temperature for 3 to 5 min (finished within 10 min). The judgment method is

shown in Fig. 3, when the control line appears, it indicates the detection is effective,

and at the same time, when only the No. 1 detection line appears, it indicates the

UL52 gene of the porcine pseudorabies virus is detected, and thus a detection result is

positive for vaccine virus; when the No. 2 detection line appears, it indicates the gE

gene of the porcine pseudorabies virus is detected, and if the No. 1 detection line also

appears, a detection result is positive for porcine pseudorabies wild virus; and if

neither of the No. 1 and 2 detection appears, a detection result is negative.

4. Specific embodiments Embodiment 1 Preparation of UL52 gene and gE gene plasmids of PRV for an experiment on the sensitivity A pMD18-T cloned plasmid was used as a carrier to construct recombinant plasmids respectively containing a UL52 gene and a gE gene of a PRV according to the conventional molecular cloning method, the recombinant plasmids were named pMD18-T-UL52 and pMD18-T-gE, respectively. Sequences of the UL52 gene and gE gene of the PRV referred to 47996-48309nt and 122529-122714nt coded sequences of a GenBank No. JF797218, respectively. An ultraviolet spectrophotometer was used to measure the concentrations of the recombinant plasmids 3 times and average values were taken, the base numbers of the two recombinant plasmids were 2915 bp and 2892 bp, respectively, the plasmid copy number was calculated according to the following formula, i.e. the copy number (copies/L)=6.02x1023xthe concentration of the plasmid (ng/L)x10-9/(the base number of the plasmidx660), and finally 3.3x1 9copies/L was used as the initial concentration. The two recombinant plasmids were respectively diluted according to 10 times gradient to obtain plasmids at 5 gradient concentrations of 3.3x10, 3.3x102, 3.3x103, 3.3x104, and 3.3x105 which were used as templates, and the plasmids were subpackaged and marked for later use. Embodiment 2 Preparation of a SmartMix RPA reaction liquid The conventional RPA reaction liquid system contains the following components: 100 ng/4L recombinase uvsX; 50 ng/4L accessory protein uvsY; 100 ng/4L LGp 32 protein (single-stranded DNA binding (SSB)); 25 ng/4L Bsu DNA polymerase; 0.5 mM dNTPs; 1 mM DTT (dithiothreitol); 3 mM ATP (adenosine triphosphate); 100 mM Tricine (trimethylglycine trimethylglycocoll) with a pH of 8.0; 5% PEG 20K (polyethylene glycol 20000); 100 g/4L Creatine kinase (creatine kinase); 20 mM Phosphokinase (phosphokinase); and 80 mM Potassium acetate (potassium acetate). units of NFO enzymes (endonuclease IV), 50 pM UL52primerl, 50 pM UL52primer2, 10 pM UL52probe, 50 pM gEprimerl, 50 pM gEprimer2, and 10 pM gEprobe were added on the basis of the conventional RPA reaction liquid, the mixture was uniformly mixed and subpackaged according to 45.5 L per tube to obtain RPA or NFO-RPA SmartMix rapid reaction liquids which were stored at a low temperature for later use. In addition, 100 mM Magnesium Acetate (magnesium acetate) was subpackaged according to 2.5 per tube and stored at room temperature for later use, which were used as RPA amplification primer fluids.

Embodiment 3 Paramagnetic particle method for extracting PRV nucleic acid of

a procine serum sample

For plasma, serum, ascites, cell culture product and other liquid samples, 200 [

of the sample was directly taken and added into a 1.5 mL nuclease-free centrifuge

tube, 500 1 of lysis solution (10 mmol/L Tris.Cl (pH 8.0), 10 mmol/L EDTA, 150

mmol/L NaCL, 0.5% SDS, and 100 to 200 g/ml protease K) was added into the tube,

and the mixture was thoroughly oscillated and uniformly mixed.

The sample mixture was lysed in a warm bath at 65 to 70°C for 5 min.

10 L of thoroughly mixed magnetic bead solution (magnetic beads were labeled

condition of high salt ion, the silicone plasmalemma binds the nucleic acid by means

of positive and negative electric adsorption force, and in a low-salt ion environment,

the nucleic acid is eluted and separated to achieve the purpose of nucleic acid

separation) was added into the centrifuge tube, and the mixture was uniformly mixed

at room temperature for 10 min by inverting the centrifuge tube.

The tube was placed on a magnetic stand for 1 min to allow the magnetic beads

in the tube to be absorbed, liquid in the tube was removed by using a pipettor, and the

centrifuge tube was removed.

500 pL of protein-removed rinse solution (50 mM Tris-HCl with a pH of 8.0, 200

mM NaCl, and 50% ethanol solution) was added into the tube to allow the magnetic

beads to be resuspended, the centrifuge tube was placed on the magnetic stand for 1

min, liquid in the tube was removed by using the pipettor, and the centrifuge tube was

removed.

500 L of washing solution (70% ethanol solution) was added into the tube to

allow the magnetic beads to be resuspended, the centrifuge tube was placed on the magnetic stand for 1 min, liquid in the tube was removed by using the pipettor, and the centrifuge tube was removed.

500 L of washing solution (the 70% ethanol solution) was added again into the

tube to allow the magnetic beads to be resuspended, the centrifuge tube was placed on

the magnetic stand for 1 min, liquid in the tube was removed by using the pipettor, at

the same time the centrifuge tube was kept on the magnetic stand to allow the

magnetic beads continued to be absorbed and were dried in the air at room

temperature for 5 min.

The centrifuge tube was removed from the magnetic stand, 50 to 100 L of

eluent solution (TE buffer solution with a pH of 8.0, 10 mM Tris-HCl, and 1mM

EDTA) was added into the tube to allow the magnetic beads to be resuspended, and

the tube was placed in a warm bath at 55C for 5 min, and was slightly shaken twice

in the warm bath to allow the nucleic acid (extracted virus genome DNA) to be

thoroughly eluted.

The centrifuge tube was placed on the magnetic stand for 1 min so that the

magnetic beads were absorbed, liquid was transferred into a new 1.5 ml RNA

Embodiment 4 Comparative experiment on the sensitivity of RPA-GICA

combined detection of a UL52 gene and a gE gene of PRV

I. Operating steps of the RPA-GICA combined detection

Step 1: the pMD18-T-UL52 and pMD18-T-gE plasmids at 5 gradient

concentrations prepared in Embodiment 1 were respectively used as detection

templates, 2 L of each template was taken and added to the numbered RPA

SmartMix prepared in Embodiment 2, and the mixture was uniformly mixed until

47.5 L of the total system was obtained and subjected to instantaneous centrifugation

for 3 s.

Step 2: 2.5 L of RPA reaction primer fluid prepared in Embodiment 2 was

respectively added into an internal opening of a cover of each sample reaction tube at

step 1, top covers were buckled up tightly in turn, the mixture was manually oscillated to 10 times, and was uniformly mixed until 50 L of the total system was obtained and subjected to instantaneous centrifugation for 3 s.

Step 3: the reaction tube was placed in a thermostatic metal bath for reacting at

38C for 20 min.

Step 4: 5 L of each reaction liquid at step 3 was respectively taken, diluted with

pL of pure water, and dropwise added to a sampling hole of a GICA test strip, and

the test strip was placed at room temperature for 5 to 10 min.

Step 5: a detection result of the test strip was read, as show in Fig. 4, and a

judgment is implemented with reference to Fig. 3. The upper limit of the RPA-GICA

detection for the UL52 gene and the gE gene of the PRV was 3.3x10 copies/L. When

the UL52 gene and gE gene plasmid templates of the PRV reached 3.3x103, No. 1 and

2 detection lines reached the maximum signal peak values, which did not increase as

the increase of the concentration of the template.

II. Operating steps of an experiment on the sensitivity of PCR detection of a

UL52 gene and a gE gene of PRV:

Step 1, sample adding: the pMD18-T-UL52 and pMD18-T-gE plasmids at 5

gradient concentrations prepared in Embodiment 1 were respectively used as

detection templates, 2 L of each template was taken and added into a reaction tube

marked with serial number, 15 L of 2xPCR Mix (containing 0.1 U/ l Taq DNA

polymerase, 400 mM dNTPs, 20 mM Tris-Cl, 3 mM MgCl2, and 100 mM KCl

mixture) was added into the reaction tube, 1 L of 10 pM UL52 forward and reverse

primers (UL52P1: 5'-GGAGTACTTTTACACGTCCCAGT-3' (shown as SEQ ID NO.

9) and UL52P2: 5'-CTCCGAGAGGCTGCCCAGGT-3' (shown as SEQ ID NO. 10))

and 1 [L of 10 pM gE forward and reverse primers (gEp1:

'-GGCTGTTTGTGCTGGCGCTG-3' (shown as SEQ ID NO. 11) and gEp2:

'-CGTCGTAGTAGTCCTCGTGC-3' (shown as SEQ ID NO. 12)) were respectively

added into the reaction tube, and each reaction tube was made up by 12 L of pure

water until 30 L of PCR reaction system was obtained.

Step 2, PCR amplification: the reaction tube at step 1 was placed in a PCR cycler,

and a PCR amplification program was set up as follows: predegeneration parameters were 94°C and 5 min; and amplification reaction parameters were as follows: degeneration parameters were 94C and 30 s; annealing parameters were 61°C and s; extension parameters were 72C and 1 min; there were 30 cycles in total; ultimate extension parameters were 72C and 10 min; and a storage parameter was

4°C.

Step 3, electrophoresis: 1.5% agarose gel was prepared, 5 L of each above PRC

reaction product was taken and subjected to electrophoresis at 100 V and 80 mA for

to 30 min.

Step 4, scanning observation of a PCR detection result: a Tanon1600 gel imaging

system was used to scan and record electrophoretic results.

Step 5, analysis of the PCR result: as shown in Figs. 5 and 6, the upper limit of

the PCR detection for the UL52 gene and gE gene plasmids of PRV was 3.3x102

copies/pL.

Conclusion: the sensitivity of the RPA-GICA detection for the PRV UL52 gene

and the gE gene is one order of magnitude higher than that of the PCR detection.

Embodiment 5 Comparative experiment on the specificity of RPA-GICA

combined detection of a PRV UL52 gene and a gE gene

I. Operating steps of the RPA-GICA combined detection

Step 1: commercial freeze-dried positive serum disease materials of attenuated

vaccine porcine pseudorabies virus (PRV, containing an HE-98 strain of gE; and

excluding a Bartha-K61 strain of gE), classical swine fever virus (CSFV, a CH-1R

strain), porcine reproductive and respiratory syndrome virus (PRRSV, a TJM-F92

strain), and porcine Japanese encephalitis virus (PJEV, an SA14-14-2 strain), and

positive serum disease materials of a porcine circovirus (PCV) and a porcine

parvovirus (PPV) which were separated in a laboratory were selected and used as

experimental materials. Nucleic acid of each virus was extracted by referring to

Embodiment 3, 2 L of the extracted nucleic acid of each virus was respectively

added to the numbered RPA SmartMix prepared in Embodiment 2, and the mixture

was uniformly mixed until 47.5 L of the total system was obtained, and subjected to

instantaneous centrifugation for 3 s.

Step 2: 2.5 L of the RPA reaction primer fluid prepared in Embodiment 2 was

step 1, top covers are buckled up tightly in turn, and the mixture was manually

oscillated 5 to 10 times, and was uniformly mixed until 50 L of the total system was

obtained and subjected to instantaneous centrifugation for 3 s.

38C for 20 min.

Step 4: 5 L of each reaction liquid at step 3 was taken, diluted with 75 L of

pure water, and dropwise added to a sampling hole of a GICA test strip, and the test

strip was placed at room temperature for 5 to 10 min.

Step 5, a detection result of the test strip was read, as show in Fig. 7, and a

judgment was implemented with reference to Fig. 3. The specificity of the RPA-GICA

detection of the amplified products of the UL52 gene and the gE gene of PRV was

high, and did not bind to the non-specificity of the nucleic acid amplified products of

other viruses. When both of the UL52 gene and the gE gene of PRV were detected,

No. 1 and 2 lines appeared at the same time, and when the PRV UL52 gene was

detected, the No. 1 line appeared.

II. Operating steps of a comparative experiment on the specificity of PCR

detection of a UL52 gene and a gE gene of PRV:

Step 1, virus nucleic acid extraction: commercial freeze-dried positive serum

disease materials of attenuated vaccine porcine pseudorabies virus (PRV, containing

an HE-98 strain of gE; and excluding a Bartha-K61 strain of gE), classical swine

fever virus (CSFV, a CH-1R strain), porcine reproductive and respiratory syndrome

virus (PRRSV, a TJM-F92 strain), and porcine Japanese encephalitis virus (PJEV, an

SA14-14-2 strain), and positive serum disease materials of a porcine circovirus (PCV)

and a porcine parvovirus (PPV) which were isolated in a laboratory were selected and

used as experimental materials, and nucleic acid of each virus was extracted by

referring to Embodiment 3 for later use.

Step 2, PCR amplification: 2 L of extracted nucleic acid of each virus was taken,

used as a detection template and respectively added into a reaction tube marked with serial number, 15 L of 2xPCR Mix (containing 0.1 U/l Taq DNA polymerase, 400 mM dNTPs, 20 mM Tris-Cl, 3 mM MgCl2, and 100 mM KCl mixture) was added into the reaction tube, 1 L of 10 pM UL52 forward and reverse primers (UL52P1:

'-GGAGTACTTTTACACGTCCCAGT-3' and UL52P2:

'-CTCCGAGAGGCTGCCCAGGT-3') and 1 L of 10 pM gE forward and reverse

primers (gEpl: 5'-GGCTGTTTGTGCTGGCGCTG-3' and gEp2:

'-CGTCGTAGTAGTCCTCGTGC-3') were respectively added into the reaction tube,

and finally each reaction tube was made up by 12 L of pure water until 30 L of

PCR reaction system was obtained. The reaction tube was placed in a PCR cycler, and

a PCR amplification program was set up as follows: predegeneration parameters were

94°C and 5 min; and amplification reaction parameters were as follows: degeneration

parameters were 94C and 30 s; annealing parameters were 61°C and 30 s; extension

parameters were 72C and 1 min; there were 30 cycles in total; ultimate extension

parameters were 72C and 10 min; and a storage parameter was 4°C.

Step 3, electrophoresis: 1.5% agarose gel was prepared, 5 L of each above PRC

to 30 min.

Step 4, scanning observation of a PCR detection result: a Tanon1600 gel imaging

system was used to scan and record electrophoretic results.

Step 5, analysis of the PCR result: as shown in Figs. 8 and 9, the specificity of

amplification of the UL52 gene and the gE gene of the PRV vaccine virus was

improved, but non-specific amplification results of other viruses were not found.

Conclusion: The specificity of the RPA-GICA detection for the UL52 gene and

the gE gene of PRV is equal to that of the PCR detection.

Embodiment 6 Comparative experiment on PRV nucleic acid molecular

detection of a clinical porcine blood sample

Comparative detection was performed on 15 serum pathological materials

collected from three pig farms which did not use PRV vaccines around Jinzhou City

by respectively using RPA-GICA and PCR to verify the repeatability and reliability of

the method of the present disclosure, and the detection efficiency was evaluated.

Results are shown in Table 1, the concordance of RPA-GICA and PCR was 100%, and

at the same time, 3 PRV positive samples and 12 negative samples were detected.

Table 1 Comparison results of RPA-GICA and qRT-PCR detection of 15 porcine

serum samples RPA-GICA PCR PRV positive PRV Tota negative 1 PRV 3 0 3 positive PRV 0 12 12 negative Total 9 12 15

*Table note: Concordance=100% is proved through a kappa concordance test.

The various embodiments described above are merely used to further explain the

present disclosure, but are not used to limit the scope of protection of the present

disclosure. All equivalent changes made based on the concepts of the present

disclosure and obvious improvements to the various technical solutions of the present

disclosure shall fall within the scope of protection of the present disclosure.

Claims

What is claimed is:

1. A kit for detecting a UL52 gene and/or a gE gene of a porcine pseudorabies

virus, comprising: reagents required by recombinase polymerase amplification for

amplifying a UL52 gene sequence and/or a gE gene sequence of the porcine

pseudorabies virus, a reagent required by a colloidal gold test strip for showing an

amplified product of the UL52 gene and/or an amplified product of the gE gene of the

porcine pseudorabies virus, and the colloidal gold test strip.

2. The kit for detecting the UL52 gene and/or the gE gene of the porcine

pseudorabies virus of claim 1, characterized in that:

the reagents required by recombinase polymerase amplification for amplifying

the UL52 gene sequence of the porcine pseudorabies virus comprise:

a first direction primer UL52primerl for the UL52 gene of the porcine

pseudorabies virus;

a middle probe UL52probe for the UL52 gene of the porcine pseudorabies

virus, the UL52 probe being connected with a first labeled molecule forward and a

protection label for preventing polymerization reversely, and comprising an artificial

base analogue which can be recognized and excised by an enzyme with DNA damage

repair activity in the middle;

a second direction primer UL52primer2 for the UL52 gene of the porcine

pseudorabies virus, the UL52 primer 2 being connected with a second labeled

molecule,

wherein the UL52primerl and the UL52primer2 are for the same direction of

the UL52 gene, and the UL52probe for the UL52 gene is located between the

UL52primerl and the UL52primer2; and/or the reagents required by recombinase

polymerase amplification for amplifying the gE gene sequence of the porcine

pseudorabies virus comprise:

a first direction primer gEprimerl for the gE gene of the porcine pseudorabies

virus; a middle probe gEprobe for the gE gene of the porcine pseudorabies virus, the gEprobe being connected with the first labeled molecule forward and the protection label for preventing polymerization reversely, and comprising the artificial base analogue which can be recognized and excised by the enzyme with DNA damage repair activity in the middle; a second direction primer gEprimer2 for the gE gene of the porcine pseudorabies virus, the gEprimer2 being connected with a third labeled molecule, wherein the gEprimerl and the gEprobe are for the same direction of the gE gene, and the gEprobe for the gE gene is located between the gEprimerl and the gEprimer2; and the second labeled molecule and the third labeled molecule are labeled molecules with different binding characteristics.

3. The kit for detecting the UL52 gene and/or the gE gene of the porcine

pseudorabies virus of claim 1 or 2, characterized in that:

the colloidal gold test strip comprises a sampling pad, a control line, a No. 1

detection line and/or No. 2 detection line;

the sampling pad comprises a first labeled molecule specific conjugate labeled

by colloidal gold particles;

specific first label molecule;

the No. 1 detection line is coated with a second labeled molecule specific

conjugate; and

the No. 2 detection line is coated with a third labeled molecule specific

conjugate.

4. The kit for detecting the UL52 gene and/or the gE gene of the porcine

pseudorabies virus of any one of claims 1 to 3, characterized in that:

the first labeled molecule is a fluorescein molecule (FAM), and the first

labeled molecule specific conjugate is an FAM antibody; the second labeled molecule is biotin, and the second labeled molecule specific conjugate is streptavidin; the third labeled molecule is digoxin, and the third labeled molecule specific conjugate is a digoxin antibody; the specific conjugate of the first labeled molecule specific conjugate is an antibody of the FAM antibody; the protection label for preventing polymerization is C3-spacer; and the artificial base analogue which can be recognized and excised by the enzyme with DNA damage repair activity is tetrahydrofuran.

5. The kit for detecting the UL52 gene and/or the gE gene of the porcine pseudorabies virus of any one of claims 1 to 4, characterized in that: a sequence of the UL52primerl is as follows: 5'-GCGCGCCATGGAGTACTTTTACACGTCCCAGTGCCC-3'; a sequence of the UL52primer 2 is as follows: 5'-[Biotin]-ACCTCGTCAACGTCAACCTGGGCAGCCTCTCGGAGCT-3'; a sequence of the UL52probe is as follows: 5'-[FAM]-GCGCTCAGCGAGGCCGAGCTCGAGTTCTACCGCTT-[THF] CTCTTCGCCTTCCTCT-[C3-spacer]-3'; a sequence of the gEprimerl is as follows: 5'-GACGACGCCCGGGCGGCTGTTTGTGCTGGCGCTGGG-3'; a sequence of the gEprobe is as follows: 5'-[FAM]-TGCTCCCGGCGCCGGGCGGCCTCGCGGCCGTTCC-[THF]-G GTGCCGACGCGGGCGC-[C3-spacer]-3'; a sequence of the gEprimer2 is as follows: 5'-[Dig]-CGTCGTAGTAGTCCTCGTGCGTGGGCAGGCTGGTGT-3', wherein Biotin represents a biotin molecule, Dig represents a digoxin molecule, FAM represents the fluorescein molecule (FAM), and THF represents a tetrahydrofuran molecule.

6. The kit for detecting the UL52 gene and/or the gE gene of the porcine

pseudorabies virus of any one of claims 1 to 5, further comprising:

an NFO enzyme, a recombinase uvsX, an accessory protein uvsY, a Gp 32

protein; a Bsu DNA polymerase, dNTPs, dithiothreitol, adenosine triphosphate,

trimethylglycine trimethylglycocoll, polyethylene glycol 20000, creatine kinase,

phosphokinase, potassium acetate and magnesium acetate.

7. A method for detecting a UL52 gene and/or a gE gene of a porcine

pseudorabies virus, the method being used for a non-diagnostic purpose, comprising:

amplifying a UL52 gene sequence and/or a gE gene sequence of the porcine

pseudorabies virus by means of recombinase polymerase amplification, and showing

an amplified product of the UL52 gene and/or an amplified product of the gE gene of

the porcine pseudorabies virus by using a colloidal gold test strip.

8. The method for detecting the UL52 gene and/or the gE gene of the porcine

pseudorabies virus of claim 7, comprising the following steps:

(1) extracting nucleic acid of a biological material which may comprise a

UL52 gene and/or a gE gene of a porcine pseudorabies virus;

(2) performing recombinase polymerase amplification on the nucleic acid

(3) showing an amplified product obtained at step (2) by using the colloidal

gold test strip of any one of claims I to 6.

9. The method for detecting the UL52 gene and/or the gE gene of the porcine

pseudorabies virus of claim 8, characterized in that step (2) comprises the following

substeps:

(i) taking 2 L of the extracted nucleic acid sample obtained at step (1) and

adding the same into a 0.2 mL centrifuge tube, and then adding 45.5 L of RPA

reaction liquid, 100 ng/L recombinase uvsX, 50 ng/L accessory protein uvsY, 100 ng/4L Gp 32 protein, 25 ng/L Bsu DNA polymerase, 0.5 mM dNTPs, 1 mM dithiothreitol, 3 mM adenosine triphosphate, 100 mM trimethylglycine trimethylglycocoll with a pH of 8.0, 5% polyethylene glycol 20000, 100 ng/L creatine kinase, 20 mM phosphokinase, and 80 mM potassium acetate into the centrifuge tube, wherein the RPA reaction liquid comprises labeled primers and labeled probes for specific nucleic acid amplification and detection, and specifically comprises the following components: 50 pM UL52primerl, 50 pM UL52primer2, 10 pM

UL52probe, 50 pM gEprimerl, 50 pM gEprimer2, 10 pM gEprobe, and 10 units of

NFP enzymes; and

(ii) uniformly mixing the above reaction liquid, adding 2.5 L of 100 mmol/L

magnesium acetate, uniformly mixing the mixture and placing the same in a

thermostatic metal bath for warm bathing at 38C for 15 to 30 min to obtain an

amplified product which is used as an amplified sample to be detected.

10. The method for detecting the UL52 gene and/or the gE gene of the porcine

pseudorabies virus of claim 8 or 9, characterized in that step (3) comprises the

following substeps:

(a) taking a colloidal gold test strip and balancing the same at room

temperature;

(b) adding a sample: taking the amplified product obtained at step (2) and

to a sample hole of the colloidal-gold test strip; and

(c) reading a result.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

sequence table

<110> Jinzhou Medical University

<120> METHOD AND KIT FOR DIFFERENTIALLY DETECTING PORCINE 2021103861

PSEUDORABIES VACCINE VIRUS AND WILD VIRUS

<160> 12

<170> SIPO Sequence Listing 1.0

<210> 1

<211> 36

<212> DNA

<213> Porcine pseudorabies virus

<400> 1

gcgcgccatg gagtactttt acacgtccca gtgccc 36

<210> 2

<211> 37

<212> DNA

<213> Porcine pseudorabies virus

<400> 2

acctcgtcaa cgtcaacctg ggcagcctct cggagct 37

<210> 3

<211> 35

<212> DNA

<213> Porcine pseudorabies virus

<400> 3

gcgctcagcg aggccgagct cgagttctac cgctt 35

<210> 4 2021103861

<211> 16

<212> DNA

<213> Porcine pseudorabies virus

<400> 4

ctcttcgcct tcctct 16

<210> 5

<211> 36

<212> DNA

<213> Porcine pseudorabies virus

<400> 5

gacgacgccc gggcggctgt ttgtgctggc gctggg 36

<210> 6

<211> 36

<212> DNA

<213> Porcine pseudorabies virus

<400> 6

cgtcgtagta gtcctcgtgc gtgggcaggc tggtgt 36

<210> 7

<211> 34

<212> DNA

<213> Porcine pseudorabies virus

<400> 7

tgctcccggc gccgggcggc ctcgcggccg ttcc 34 2021103861

<210> 8

<211> 17

<212> DNA

<213> Porcine pseudorabies virus

<400> 8

ggtgccgacg cgggcgc 17

<210> 9

<211> 23

<212> DNA

<213> Porcine pseudorabies virus

<400> 9

ggagtacttt tacacgtccc agt 23

<210> 10

<211> 20

<212> DNA

<213> Porcine pseudorabies virus

<400> 10

ctccgagagg ctgcccaggt 20

<210> 11

<211> 20

<212> DNA

<213> Porcine pseudorabies virus 2021103861

<400> 11

ggctgtttgt gctggcgctg 20

<210> 12

<211> 20

<212> DNA

<213> Porcine pseudorabies virus

<400> 12

cgtcgtagta gtcctcgtgc 20

附图