CN117625838A - Colloidal gold RAA method nucleic acid detection kit for cat parvovirus and detection method thereof - Google Patents

Abstract

The invention provides a colloidal gold RAA method nucleic acid detection kit and a detection method thereof, which are suitable for a colloidal gold RAA method and are designed aiming at FPV VP2 genes, wherein a specific primer probe combination suitable for the colloidal gold RAA method is designed for the FPV VP2 genes, the FPV VP2 genes are amplified by an nfo method recombinase amplification system, the nfo, uvsY and ssb protein contents in the recombinase system are optimized, the problem of nonspecific amplification in the colloidal gold RAA method detection process of the cat parvovirus FPV VP2 genes can be effectively solved, the detection time is shorter, the amplification and detection time only needs 20-30 minutes, the sensitivity is high, the sample of 1.25copies/uL can be detected at the minimum, no instrument dependence exists, the reaction can be carried out at 20-42 ℃, and after the reaction, a colloidal gold test strip can be used for judging whether the cat parvovirus infection is detected by the cat parvovirus, the detection method can be applied to a hospital, the detection at home and the home can also be carried out in real time, and the application prospect is good.

Description

Colloidal gold RAA method nucleic acid detection kit for cat parvovirus and detection method thereof

Technical Field

The invention belongs to the technical field of molecular diagnosis biology, relates to a colloidal gold RAA method nucleic acid detection kit and a detection method thereof for cat parvovirus, and particularly relates to a colloidal gold RAA method nucleic acid detection kit and a detection method thereof for cat parvovirus, which can effectively solve the problem of nonspecific amplification.

Background

Cat fever is also called cat panleukopenia, cat infectious enteritis or cat blast, a multiple infectious disease caused by cat parvovirus (Feline panleukopenia virus, FPV) can cause cat anorexia, vomiting and diarrhea, and leucocyte is severely reduced, and symptoms such as bloody stool, dehydration and failure of each organ can be caused when serious. Cat parvovirus can survive for one year in the environment, so that cats are easily exposed to the environment with the virus to be infected, especially the immunized or partially immunized kittens are more easily suffering from the disease than adult cats, the illness is more serious, the death rate is higher, the infection rate of cats under 1 year old can reach 70%, the death rate is 50-60%, and the death rate of kittens under 5 months old can reach 80-90%; pregnant kittens, if infected with the virus, can cause reproductive dysfunction, leading to abortions or severe damage to the lower cerebellum. If a healthy cat is exposed to an environment contaminated with viruses, it is also possible that the cat plague may be infected with tableware or the like used by the diseased cat, and certain blood sucking insects such as mosquitoes, wasps and lice may also be able to transmit the disease.

Cat parvovirus (FPV) is a member of the parvoviridae, bovine canine parvovirus, a single-stranded, non-enveloped DNA virus. FPV is mainly infected by various animals such as felines and weasel, and is the virus with the widest infection range and the strongest pathogenicity in the meat animal parvovirus at present. The main diagnostic methods of FPV are mainly serological methods (colloidal gold method) and virus isolation and identification and PCR methods. The sensitivity of the colloidal gold and virus separation method is low, and early infection cannot be diagnosed; the PCR method and the real-time fluorescence quantitative PCR method have high sensitivity and strong specificity, but the method has higher requirements on technicians, expensive equipment, high detection cost depending on a PCR instrument and various matched equipment and limited application range. Therefore, a rapid nucleic acid detection method with high sensitivity, convenience, rapidness and strong specificity is urgently needed, particularly a rapid nucleic acid detection method for cat parvovirus, the possibility of missed detection and false detection is avoided, and real-time test can be performed without depending on a detection instrument.

The application range of the PCR technology is widened again due to the in-vitro amplification technology of the constant temperature nucleic acid, so that the operation is more convenient. The existing isothermal nucleic acid in-vitro amplification methods mainly comprise loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA) and recombinase-mediated amplification (RAA). The recombinase mediated amplification (RAA) is a isothermal nucleic acid rapid amplification technique, which uses a recombinase obtained from bacteria or fungi, which can be tightly combined with a primer at normal temperature to form a polymer of the enzyme and the primer, and when the primer searches for a complementary sequence perfectly matched with the primer on a template DNA, the double-stranded structure of the template DNA is opened with the aid of a single-stranded DNA binding protein, and a new complementary strand of DNA is formed with the aid of a DNA polymerase, and the primer and a probe for modifying a fluorescent group are specifically bound to the complementary sequence while the double-stranded structure of the template DNA is opened. The colloidal gold RAA method isothermal amplification technology, namely, the colloidal gold is utilized to detect the amplified product, so that the convenience of detection can be remarkably improved, and the instant diagnosis is realized without depending on instruments.

CN114457197a provides a method for detecting cat parvovirus by using RAA fluorescence, which needs to detect on a fluorescence PCR amplification instrument, has long detection time, depends on the detection instrument, has low detection sensitivity, can reach only 10copies/uL, and is easy to generate false negative under the condition of low virus content, thereby causing missed detection and false detection.

Therefore, the cat parvovirus is detected by a colloidal gold RAA method isothermal amplification technology which has higher sensitivity and can be detected rapidly without depending on an instrument, and the detection process is more convenient and faster.

Disclosure of Invention

In order to solve the problems, the invention provides a colloidal gold RAA method nucleic acid detection kit and a detection method thereof, which are suitable for the colloidal gold RAA method and are designed for the FPV VP2 gene, the contents of nfo, uvsY and ssb proteins in a recombinase system are optimized, the problem of nonspecific amplification in the colloidal gold RAA method detection process of the cat parvovirus FPV VP2 gene can be effectively solved, the detection time is shorter, the amplification and detection time only needs 20-30 minutes, the sensitivity is high, 1.25copies/uL (12.5 copies/test) samples can be detected at the minimum, no instrument dependence exists, the reaction can be carried out at 20-42 ℃, and after the reaction is finished, whether the cat parvovirus infection is detected by using a colloidal gold test strip can be judged, so that the kit can be applied to the detection of a pet hospital and can be detected at home, and has good application prospects.

The colloidal gold RAA method adopted by the invention is used for detecting the target gene by using an nfo method recombinase amplification system, and the target gene is detected in real time by using a colloidal gold test strip embedded with a fluorescent group antibody and streptavidin.

However, in the colloidal gold RAA detection process of the FPV VP2 gene of the cat parvovirus, non-specific amplification is easy to occur, so that false positive occurs, and false detection is caused, which is a problem which is difficult to solve in the past. According to the invention, by designing and screening the most suitable primer probes and optimizing a recombinase system, the problem of nonspecific amplification in the detection process of the cat parvovirus FPV VP2 gene by a colloidal gold RAA method can be effectively prevented.

The invention adopts a recombinase system of a colloidal gold RAA method, which contains exonuclease (nfo protein), the nfo protein can recognize and cleave a tetrahydrofuran bond modified in the middle of a probe sequence, the sequence after the tetrahydrofuran bond of the probe is sheared, and a new amplification primer pair is formed by the 5' -end sequence of the probe and a reverse primer. The 5 'end of the probe is modified with FAM fluorescent groups, the 5' end of the reverse primer is modified with biotin, so that the 5 'end of the amplified DNA fragment carries FAM fluorescent groups, the 3' end carries biotin groups, amplification is carried out for 10-15min, and then the amplification product is detected by using a strip gold test strip embedded with FAM antibodies and streptavidin, so that whether the sample contains cat parvovirus can be judged.

In one aspect, the invention provides a colloidal gold RAA method nucleic acid detection primer probe group of a cat parvovirus FPV VP2 gene, comprising:

(1) A forward primer having a sequence as shown in SEQ ID NO. 1;

(2) A reverse primer having a sequence shown as SEQ ID NO.2, and modified biotin at the 5' -end;

(3) The probe has a sequence shown as SEQ ID NO.3, a fluorescent group is modified at the 5 'end and is subjected to blocking modification at the 3' end, wherein the fluorescent group is any one selected from FAM, CY5 and ROX.

Further, FAM fluorescent groups are modified at the 5 'end of the probe, phosphorylation modification is carried out at the 3' end, and tetrahydrofuran is modified at the 30 th-33 th base.

Unlike other isothermal amplified nucleic acid techniques, the colloidal gold RAA method requires that nfo protein recognize and cleave the intermediate modified tetrahydrofuran bond of the probe sequence, the probe tetrahydrofuran bond is cleaved off, and the 5' sequence of the probe and the reverse primer together form a new amplification primer pair. Therefore, the design of the primer probe is completely different from other isothermal amplification technologies, the primer probe is directly related to reaction specificity and sensitivity, and the length of the primer is longer, so that the identification and the specificity of a chain in the amplification process are ensured, and the non-specific amplification is prevented. Because the primer is longer, a secondary structure is easier to form, and the primer dimer is amplified at 37-42 ℃, the secondary structure is not easy to open, and the amplification efficiency is affected, so the design difficulty is higher.

The invention designs a plurality of pairs of primer probes aiming at the FPV VP2 gene, selects the primer probe combination which is most suitable for the colloidal gold RAA method and can obviously improve the detection sensitivity and the specificity, thereby effectively preventing the nonspecific amplification; the primer probe combination is adopted to detect the FPV VP2 gene by a colloidal gold RAA method, the sensitivity is high, a sample of 1.25copies/uL (12.5 copies/test) can be detected at the lowest, and the amplification and detection time only needs 20-30 minutes.

On the other hand, the invention provides a colloidal gold RAA method nucleic acid detection kit of the cat parvovirus FPV VP2 gene, which comprises the primer probe set.

Further, a dry recombinase enzyme powder comprising nfo protein is also included.

Further, the recombinant enzyme dry powder also contains uvsX protein, uvsY protein, DNA polymerase P and ssb protein.

Further, the recombinant enzyme dry powder comprises 1 μg-2.5 μg of uvsX protein, 0.5 μg-2.5 μg of uvsY protein, 0.5 μg-2.5 μg of DNA polymerase P,1.5 μg-5 μg of ssb protein, and 0.5 μg-3 μg of nfo protein.

Further, the recombinant enzyme dry powder shown comprises 1.0. Mu.g of uvsX protein, 1.0. Mu.g of uvsY protein, 1.5. Mu.g of DNA polymerase P, 2.5. Mu.g of ssb protein, 0.5. Mu.g of nfo protein.

The RAA recombinase dry powder tube is freeze-dried powder of various enzymes, and the target fragment is amplified at 25-42 ℃ by adding reaction liquid. Wherein, the nfo protein can recognize and enzyme-cut tetrahydrofuran bond modified in the middle of the probe sequence, cut the probe, form primer pairs by 5' end fragment and reverse primer of the probe, amplify and obtain DNA fragment; the DNA polymerase P is derived from DNA polymerase I of escherichia coli, and specific extension is realized on the DNA after chain replacement; the ssb protein is a single-chain binding protein, the Uvs X protein is a homologous recombinant protein, the Uvs X, uvs Y and ssb proteins mediate melting of the template and enable strand replacement between the template and the primer, which requires ATP to provide energy.

The invention finds that the proportion and concentration of each protein in the recombinase dry powder system are critical for the sensitivity and specificity of the reaction, in particular nfo protein for identifying and cleaving the probe, ssb protein and uvsY protein which mediate melting of the template and realize chain replacement between the template and the primer.

A large number of researches prove that in the RAA recombinase dry powder system, the detection result of the FPV VP2 gene is better and the strip is clearer by optimizing the contents of nfo protein, uvsY protein and ssb protein. The reason is that the nfo protein is too high in the FPV VP2 gene detection process, so that non-specific amplification is caused, false positive occurs, and false detection is caused; the increase of the uvsY protein and ssb protein content can improve the recombination amplification efficiency and is beneficial to improving the detection sensitivity of the FPV VP 2.

Further, the kit also comprises a reaction Buffer A, a Buffer B, a negative control, a positive control and a colloidal gold test strip, wherein FAM antibodies and biotin antibodies are embedded on the colloidal gold test strip.

Further, the reaction Buffer a is: 10-30mM Tris-HCl (pH 7.5), 0.1mM-0.5mM dNTP, 3% -9% PEG8000 or PEG of other molecular weight, 1-5mM DTT,10-20mM ATP,10-50mM phosphoenolpyruvate, and 500-1500 ng/. Mu.L pyruvate kinase.

Further, the Buffer B:2-10mM Mg ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the The positive control is a pseudo virus containing FPV Vp2 gene fragments, and the Seq ID No.4; the negative control was physiological saline.

The reaction Buffer A is used for maintaining the pH value of a reaction system, providing salt ions and stable pH for the reaction, and affecting the activity of RAA enzyme; providing a raw material for DNA synthesis, and energy. Buffer B is an activator of the reaction, and a proper amount of Buffer B is added to start the reaction.

In yet another aspect, the present invention provides a method for carrying out the detection of the feline parvovirus FPV VP2 gene using the kit as described above, said method comprising the steps of:

(1) Extracting nucleic acid to be detected, and preparing positive control and negative control;

(2) Preparing recombinant enzyme dry powder, placing the recombinant enzyme dry powder in a reaction tube, and adding a reaction Buffer A, an upstream primer, a downstream primer and a fluorescent probe;

(3) Adding nucleic acid to be detected, positive control and negative control into a reaction tube, adding Buffer B, shaking, mixing uniformly, and centrifuging;

(4) Isothermal amplification for 5-15min;

(5) Adding 5-10 times of diluent into the amplified product, then detecting by using a colloidal gold test strip, and judging the detection result.

The reason why the amplification product needs to be diluted with 5-10-fold dilution is that: if the amplification product is directly used for chromatography, the chromatography speed is very slow, and the detection effect is affected.

The beneficial effects of the invention are as follows:

1. by utilizing nfo exonuclease to cut a probe, amplifying a nucleic acid fragment with a fluorescent modification group at the 5 'end and biotin modification at the 3' end, displaying an amplified product by using a colloidal gold test strip, and realizing the instantaneity of detection without depending on a detection instrument;

2. the primer probe group most suitable for detecting the VP2 gene of the cat parvovirus by utilizing the recombinase dry powder is found out, the contents of nfo, uvsY and ssb proteins in a reaction system are optimized, the problem of nonspecific amplification is effectively solved, a sample of 1.25copies/uL can be detected at the lowest, the amplification and detection time is only required to be 20-30 minutes, the detection speed is high, the sensitivity is high, and the specificity is strong.

3. The method is simple to operate, does not depend on a precise nucleic acid amplification instrument and a detection technology, is short in detection time and high in sensitivity, is widely suitable for quick, effective and economic detection requirements of customers, can be transported at normal temperature, is suitable for a large number of scenes, can be used in pet hospitals, can be used for home detection, and has a wide application prospect.

Drawings

FIG. 1 is a detection result of the primer probe set 1 in example 3, wherein A: positive control, 100copies/test; 50copies/test, D negative control;

FIG. 2 shows the detection results of the primer probe set 2 in example 3, wherein A: positive control, 100copies/test; 50copies/test, D negative control;

FIG. 3 is a detection result of the primer probe set 3 in example 3, wherein A: positive control, 100copies/test; 50copies/test, D negative control;

FIG. 4 shows the results of the detection of group 1 recombinant enzyme dry powders in example 4, A: positive control, 100copies/test; 50copies/test, D negative control;

FIG. 5 shows the results of the detection of the group 2 recombinant enzyme dry powder of example 4, A: positive control, 100copies/test; 50copies/test, D negative control;

FIG. 6 shows the results of the detection of group 3 recombinant enzyme dry powders in example 4, A: positive control, 100copies/test; 50copies/test, D negative control;

FIG. 7 shows the results of the assay for group 1 recombinant enzyme dry powders of example 5, wherein A is a positive control, B is 100copies/test, C is 50copies/test, and D is a negative control;

FIG. 8 shows the results of the detection of the dry powder of the recombinant enzyme of group 2 in example 5, wherein A is a positive control, B is 100copies/test, C is 50copies/test, and D is a negative control;

FIG. 9 is a sample test result of 100copies/test, 50copies/test in example 6, wherein A:100copies/test; b50 copies/test; negative control;

FIG. 10 is a sample test result of 25copies/test, 12.5copies/test in example 6, wherein A is 25copies/test; b12.5 copies/test; negative control.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto. The reagents not specifically mentioned in this example are all known products and are obtained by purchasing commercially available products.

Example 1 expression of nfo protein

In this example, gene synthesis was carried out by Shanghai worker on the nfo endonuclease IV gene (GeneID: 946669) of Escherichia coli, and the nfo protein was obtained by expression and purification in Escherichia coli using the nfo gene.

Example 2A nucleic acid detection kit for feline parvovirus and detection method thereof

In this example, using the nfo protein provided in example 1, a kit was prepared with the following specific composition:

1. RAA enzyme dry powder tube: 1.0 μg-2.5 μg (corresponding to 20 ng/. Mu.L-50 ng/. Mu.L) (preferred 1 μg in this example) of the uvsX protein (Hangzhou grass rider, lot: X20211018), 0.5 μg-2.5 μg (corresponding to 10 ng/. Mu.L-50 ng/. Mu.L) (preferred 1 μg in this example) of the uvsY (Hangzhou grass rider, lot: Y20211022), 0.5 μg-2.5 μg (corresponding to 10 ng/. Mu.L-50 ng/. Mu.L) (preferred 1.5 μg in this example) of the DNA polymerase P (Hangzhou grass rider, lot: P20211014) and 1.5 μg-5 μg (corresponding to 30 ng/. Mu.L-100 ng/. Mu.L) (preferred 2.5 μg in this example) of the ssb protein (Hangzhou grass rider, lot: S20211028), 0.5 μg-3.0 μg (corresponding to 20 ng/. Mu.L-50 ng/. Mu.L) (preferred 1.5 ng/. Mu.L in this example) of the ssb protein (preferred 0 ng/. Mu.L-40 ng/. Mu.L);

2. reaction Buffer a:10-30mM Tris-HCl (pH 7.5), 0.1mM-0.5mM dNTP, 3-9% PEG8000 or PEG of other molecular weight, 1-5mM DTT,10-20mM ATP,10-50mM phosphoenolpyruvate, 500-1500 ng/. Mu.l pyruvate kinase;

3. activator (Buffer B): 2-10mM Mg ²⁺ ；

4. Primer probe group: aiming at the virus to be detected, corresponding upstream primer, downstream primer and probe are designed, and the proportion is as follows: 0.5-2. Mu.L of 10mM upstream primer FPV-FP (SEQ ID NO. 1), 0.5-2. Mu.L of 10mM downstream primer FPV-RP (SEQ ID NO. 2), 0.5-1. Mu.L of 10mM probe FPV-P (SEQ ID NO. 3);

5. positive control: pseudoviruses containing FPV VP2 gene fragments have a sequence shown as SEQ ID NO.4;

6. the negative control was normal saline.

7. Colloidal gold test strip: a strip of gold test paper containing FAM antibody and streptavidin (Hexagon Biotechnology Co., ltd., lot: 40009).

The method for carrying out rapid nucleic acid detection of the feline parvovirus by using the kit comprises the following steps:

(1) Extracting nucleic acid, namely extracting according to the specification of a virus nucleic acid extraction kit, and extracting positive control and negative control at the same time;

(2) 10. Mu.L of RAA reaction Buffer A, 1. Mu.L of 10mM upstream primer, 1. Mu.L of 10mM downstream primer, and 0.5. Mu.L of 10mM probe were added to the RAA enzyme dry powder tube;

(3) Into the reaction tube was added 5. Mu.L of the coreThe product was extracted with acid, then 2.5. Mu.L Buffer B was added, followed by 30. Mu.L ddH ₂ Supplementing the reaction system to 50 mu L, vibrating and mixing uniformly, and rapidly centrifuging for 5-10S;

(4) Placing the reaction tube into a PCR amplification instrument or a water bath kettle, reacting for 5-15 minutes at 25-42 ℃, and adding 5-10 times of reaction diluent after the reaction is finished;

(5) Inserting a test strip into the diluted reaction liquid for 5-15min, and judging whether the sample is positive according to the indication belt; the judgment method is shown in Table 1.

TABLE 1 test strip test result determination

Detection result	Result determination
		Only one C line	Negative of
Two lines (C line and T line)	Positive and negative
		Without straps	Reaction is ineffective and retest is required
C line has no strip, T line has strip	Requiring retesting

Example 3 primer set screening for detecting feline parvovirus by colloidal gold RAA method

Unlike other isothermal amplified nucleic acid techniques, the colloidal gold RAA method requires that nfo protein recognize and cleave the intermediate modified tetrahydrofuran bond of the probe sequence, the probe tetrahydrofuran bond is cleaved off, and the 5' sequence of the probe and the reverse primer together form a new amplification primer pair. Therefore, the design of the primer probe is completely different from other isothermal amplification technologies, and the length of the primer is longer, so that the identification and the specificity of the chain in the amplification process are ensured. Because the primer is longer, a secondary structure is easier to form, and the primer dimer is amplified at 37-42 ℃, the secondary structure is not easy to open, and the amplification efficiency is affected, so the design difficulty is higher.

At present, no primer probe design software aiming at the colloidal gold RAA method exists, and wide screening is needed. The design of a primer probe for the recombinase-mediated amplification detected by a colloidal gold method is also unique, the 5' end of the probe sequence is modified by a fluorescent group, the middle is connected by a tetrahydrofuran bond, the 3' end is modified by phosphorylation, and the 5' end of the reverse primer is modified by biotin; in isothermal amplification, nfo exonuclease recognizes the tetrahydrofuran bond first, cleaves the probe, allowing the sequence of the probe after the tetrahydrofuran bond to be cut off, and the remaining 5' end sequence of the probe forms a new amplification primer pair with the reverse primer, the forward primer being used to increase the concentration of nucleic acid template in the sample. The DNA fragment with fluorescent groups at the 5 'end and biotin at the 3' end can be obtained through the amplification of the novel primer pair.

The kit and the detection method provided in example 2 are used in this example to detect FPV, wherein the composition of the recombinase dry powder is: 1.0. Mu.g of novel uvsX, 1.0. Mu.g of uvsY, 1.5. Mu.g of DNA polymerase P and 2.5. Mu.g of ssb protein, 0.5. Mu.g of nfo protein, reaction Buffer A:20mM Tris-HCl (pH 7.5), 0.15mM dNTP, mass percent 9%PEG8000,1mM DTT,10mM ATP,20mM phosphoenolpyruvate, 500 ng/. Mu.l pyruvate kinase; activator (Buffer B): 2mM Mg ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the And a plurality of primer probe sets are designed for VP2 gene of FPV, three sets listed in Table 2, namely, set 1, set 2 and set 3 are selected, and then synthesis is carried out on Shanghai creatures, and the primer probes are diluted to 10mM.

TABLE 2 primer probe set designed for cat parvovirus VP2 Gene

The synthesized pseudoviruses were diluted with physiological saline at various concentration gradients (0, 5, 10 copies/. Mu.L) and then extracted using the purchased viral nucleic acid extraction kit [ Tiangen Biochemical technology (Beijing), virus genome DNA/RNA extraction kit (DP 315)]According to the instructions to 200u L sample for nucleic acid extraction, using 100u L RNase-Free ddH ₂ O was eluted, and then 5. Mu.L of the eluted product was taken for rapid nucleic acid detection.

The reaction liquid system is as follows:

the test results are shown in FIGS. 1 to 3, respectively, and FIG. 1 shows the detection results of the primer probe set 1, wherein A: positive control, 100copies/test; 50copies/test, D negative control; fig. 2 shows the detection result of the primer probe set 2, wherein a: positive control, 100copies/test; 50copies/test, D negative control; FIG. 3 shows the detection results of primer probe set 3, wherein A: positive control, 100copies/test; c50 copies/test, D negative control.

As can be seen from FIGS. 1 to 3, the primer probe of the set 1 has strong specificity and clear detection results, and the negative controls of the sets 2 and 3 have nonspecific bands, so that nonspecific amplification can be effectively prevented only by selecting the set 1 as the primer probe set of the kit.

Injection of copy number of reaction = sample concentration sample amount/elution volume amplified template amount

Example 4 Effect of the amount of nfo protein on non-specific amplification

The kit provided in this example was according to example 2, and the nfo content in the RAA enzyme dry powder tube was optimized to be 0.3, 0.5, and 1 μg, respectively, and the specific recombinase dry powder was configured as follows:

group 1: 1. Mu.g of uvsX protein, 1.0. Mu.g of uvsY protein, 1.5. Mu.g of DNA polymerase P and 2.5. Mu.g of ssb protein, 0.3. Mu.g of nfo protein;

group 2: 1. Mu.g of uvsX protein, 1.0. Mu.g of uvsY protein, 1.5. Mu.g of DNA polymerase P and 2.5. Mu.g of ssb protein, 0.5. Mu.g of nfo protein;

group 3: 1. Mu.g of uvsX protein, 1.0. Mu.g of uvsY protein, 1.5. Mu.g of DNA polymerase P and 2.5. Mu.g of ssb protein, 1. Mu.g of nfo protein;

the synthesized pseudoviruses were diluted with physiological saline at various concentration gradients (10, 5, 0 copies/. Mu.L) and then extracted using the purchased viral nucleic acid extraction kit [ Tiangen Biochemical technology (Beijing), virus genome DNA/RNA extraction kit (DP 315)]According to the instructions to 200u L sample for nucleic acid extraction, using 100u L RNase-Free ddH ₂ O was eluted, and then 5. Mu.L of the eluted product was taken for rapid nucleic acid detection. The reaction liquid system and the reaction procedure are as shown in example 3.

The detection results are shown in fig. 4, 5 and 6, wherein fig. 4 shows the detection results when the dry powder of the 1 st group of recombinant enzymes is adopted, fig. 5 shows the detection results when the dry powder of the 2 nd group of recombinant enzymes is adopted, and fig. 6 shows the detection results when the dry powder of the 3 rd group of recombinant enzymes is adopted.

As can be seen from FIGS. 4 to 6, the specificity of the recombinant enzyme dry powder of group 2 is very good, the detection result is clear, the detection sensitivity of group 1 is reduced, the detection band of group 3 is weaker, and the non-specific band appears in the negative control of group 3, so that the specificity of the recombinant enzyme dry powder amplification of 0.5 mug nfo protein of group 2 is the highest, the band is the most clear, the non-specific amplification can be effectively prevented, and the reason may be that the amplification efficiency is affected by the excessively high or excessively low content of nfo protein.

Example 5 Effect of Uvs Y and ssb protein content on improving the detection sensitivity of FPV VP2 Gene

The kit provided in this example according to example 2, while varying the uvsY and ssb protein content in the RAA enzyme dry powder tube, the specific recombinase dry powder configuration is as follows:

group 1: 1. Mu.g of uvsX protein, 0.5. Mu.g of uvsY protein, 1.5. Mu.g of DNA polymerase P and 1.5. Mu.g of ssb protein, 0.5. Mu.g of nfo protein;

group 2: 1. Mu.g of uvsX protein, 1.0. Mu.g of uvsY protein, 1.5. Mu.g of DNA polymerase P and 2.5. Mu.g of ssb protein, 0.5. Mu.g of nfo protein.

The synthesized pseudoviruses were diluted with physiological saline at various concentration gradients (10, 5, 0 copies/. Mu.L) and then extracted using the purchased viral nucleic acid extraction kit [ Tiangen Biochemical technology (Beijing), virus genome DNA/RNA extraction kit (DP 315)]According to the instructions to 200u L sample for nucleic acid extraction, using 100u L RNase-Free ddH ₂ O was eluted, and then 5. Mu.L of the eluted product was taken for rapid nucleic acid detection. The reaction liquid system and the reaction procedure are as shown in example 3.

The detection results are shown in FIGS. 7 and 8, and FIG. 7 shows the detection results when the group 1 recombinase dry powder is used, wherein A is positive control, B is 100copies/test, C is 50copies/test, and D is negative control; FIG. 8 shows the results of the detection using the group 2 recombinase dry powder, wherein A is a positive control, B is 100copies/test, C is 50copies/test, and D is a negative control.

As can be seen from FIGS. 7 to 8, the detection result is better and the band is clearer when the recombinant enzyme dry powder of group 2 is detected. The contents of ssb protein and UvsY protein are improved, and the detection sensitivity is obviously improved. The reason is that the high content of the uvsY protein and the ssb protein can improve the recombination amplification efficiency, is beneficial to improving the detection sensitivity of the FPV VP2, and can influence the recombination amplification effect due to the fact that the content of the uvsY protein and the ssb protein is too high.

Example 6 Effect of different concentration gradient samples on detection

The kit and the detection method provided in the example 2 are adopted in the example to detect the cat parvovirus (FPV) VP2 gene, wherein the composition of the recombinase dry powder is as follows: 1. Mu.g of uvsX protein, 1.0. Mu.g of uvsY, 1.5. Mu.g of DNA polymerase P and 2.5. Mu.g of ssb protein, 0.5. Mu.g of nfo protein, the pseudoviruses synthesized were diluted with physiological saline in different concentration gradients: 10copies/uL, 5copies/uL, 2.5copies/uL, 1.25copies/uL, then extracted with the purchased viral nucleic acid extraction kit [ Tiangen Biochemical technology (Beijing) Co., ltd., viral genome DNA/RNA extraction kit (DP 315)]According to the instructions to 200u L sample for nucleic acid extraction, using 100u L RNase-Free ddH ₂ O is eluted, then 5 mu L of eluted product is taken for rapid reactionAnd (5) detecting nucleic acid. The reaction liquid system and the reaction procedure are as shown in example 2. The detection results are shown in fig. 9 and 10, and fig. 9 shows sample detection results of 100copies/test and 50copies/test, wherein a:100copies/test; b50 copies/test; negative control; FIG. 10 is a sample test result of 25copies/test, 12.5copies/test, wherein A is 25copies/test; b12.5 copies/test; negative control. As can be seen from FIG. 10, at a concentration of 12.5copies/test, it can still be effectively detected.

1.25copies/uL sample described in this example, 200uL sample was extracted, eluted with 100uL of eluent, and then 5uL of DNA template was amplified, and sensitivity was calculated: 1.25copies/uL 200uL/100uL 5=12.5 copies/test, it can be seen that the sensitivity of the kit provided by the invention for detecting cat parvovirus (FPV) VP2 gene can reach 1.25copies/uL or 12.5copies/test.

While the invention has been described in detail in the foregoing general description and specific embodiments, the above embodiments are illustrative, and specific features, structures, materials, or characteristics described may be combined or combined in any suitable manner in any one or more embodiments, and modifications or improvements may be made thereto as will be apparent to those skilled in the art. Accordingly, such modifications, adaptations, substitutions, or alternatives can be made without departing from the spirit of the invention and are intended to be within the scope of the invention as set forth in the following claims.

Claims (10)

1. A colloidal gold RAA method nucleic acid detection primer probe group of a cat parvovirus FPV VP2 gene is characterized by comprising the following steps:

(1) A forward primer having a sequence as shown in SEQ ID NO. 1;

(2) A reverse primer having a sequence shown as SEQ ID NO.2, and modified biotin at the 5' -end;

(3) The probe has a sequence shown as SEQ ID NO.3, a fluorescent group is modified at the 5 'end and is subjected to blocking modification at the 3' end, wherein the fluorescent group is any one selected from FAM, CY5 and ROX.

2. The primer probe set of claim 1, wherein the probe has a FAM fluorophore modified at the 5 'end and a tetrahydrofuran modified at the 3' end, and wherein the tetrahydrofuran is modified at the 33 th base.

3. A colloidal gold RAA method nucleic acid detection kit for a cat parvovirus FPV VP2 gene, comprising the primer probe set of claim 1.

4. The kit of claim 3, further comprising a dry recombinase enzyme powder comprising nfo protein.

5. The kit of claim 4, wherein the dry powder of recombinant enzyme further comprises uvsX protein, uvsY protein, DNA polymerase P, and ssb protein.

6. The kit of claim 5, wherein the dry powder of recombinase enzyme comprises 1 μg to 2.5 μg of uvsX protein, 0.5 μg to 2.5 μg of uvsY protein, 0.5 μg to 2.5 μg of DNA polymerase P,1.5 μg to 5 μg of ssb protein, and 0.5 μg to 3 μg of nfo protein.

7. The kit of claim 6, wherein the recombinase dry powder comprises 1.0 μg of uvsX protein, 1.0 μg of uvsY protein, 1.5 μg of DNA polymerase P,2.5 μg of ssb protein, and 0.5 μg of nfo protein.

8. The kit of claim 7, further comprising reaction Buffer a, buffer B, negative control, positive control, and a colloidal gold test strip having FAM antibody and streptavidin embedded thereon.

9. The kit of claim 8, wherein the reaction Buffer a is: 10-30mM Tris-HCl (pH 7.5), 0.1mM-0.5mM dNTP, 3% -9% PEG8000 or PEG of other molecular weight, 1-5mM DTT,10-20mM ATP,10-50mM phosphoenolpyruvate, and 500-1500 ng/. Mu.L pyruvate kinase; the Buffer B:2-10mM Mg ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the The positive control is a pseudo virus containing FPV Vp2 gene fragments, and the Seq ID No.4; the negative control was physiological saline.

10. A method for carrying out the detection of the feline parvovirus FPV VP2 gene using the kit according to any one of claims 3 to 9, characterized by comprising the steps of:

(1) Extracting nucleic acid to be detected, and preparing positive control and negative control;

(2) Preparing recombinant enzyme dry powder, placing the recombinant enzyme dry powder in a reaction tube, and adding a reaction Buffer A, an upstream primer, a downstream primer and a fluorescent probe;

(3) Adding nucleic acid to be detected, positive control and negative control into a reaction tube, adding Buffer B, shaking, mixing uniformly, and centrifuging;

(4) Isothermal amplification for 5-15min;

(5) Adding 5-10 times of diluent into the amplified product, then detecting by using a colloidal gold test strip, and judging the detection result.