CN115161414A

CN115161414A - Specificity detection target of monkeypox virus, oligonucleotide and kit thereof

Info

Publication number: CN115161414A
Application number: CN202210656992.8A
Authority: CN
Inventors: 张太松; 焦明超; 陈斐斐; 刘学锋
Original assignee: Acon Biotech Hangzhou Co Ltd
Current assignee: Acon Biotech Hangzhou Co Ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-10-11
Also published as: WO2023236563A1

Abstract

The invention relates to a specificity detection target of a monkeypox virus, and oligonucleotide, a kit and a method for detecting the monkeypox virus, wherein the specificity detection target comprises a primer and a probe which are designed aiming at an F3L gene region in a monkeypox virus genome, and nucleic acid of the monkeypox virus is detected by adopting a fluorescence PCR technology after nucleic acid in a sample is rapidly extracted; the method is simple to operate, and can specifically distinguish the vaccinia virus with high sequence similarity by adopting the TaqMan probe to detect the monkeypox virus target gene. False detection and missed detection can be avoided.

Description

Specificity detection target of monkeypox virus, oligonucleotide and kit thereof

Technical Field

The invention belongs to the fields of life science and biotechnology, and particularly relates to a specificity detection target of monkeypox virus, oligonucleotide, a method and a kit for detecting monkeypox virus, wherein the oligonucleotide, the method and the kit are used for detecting monkeypox virus nucleic acid in a clinical sample by adopting a fluorescence PCR technology.

Background

Monkeypox virus (monkeypoxx virus) is a "close relative" to variola virus which has been heavily abused by humans for thousands of years in history. Monkeypox virus was first discovered in 1958, when a group of monkeys used in the study presented "pox" infections, hence its name. Since the world health organization announced in 1980 that humans eradicated smallpox, monkeypox virus has become an orthopoxvirus with the greatest impact on public health. Since 2022, day 5 and day 13, 92 diagnosed cases and 28 suspected cases were reported in countries where monkeypox virus had not been circulating before the world, and there is a possibility that more cases will be found in these and other countries in the future. Currently, diagnosed and suspected cases of monkeypox are mainly from british, spain and portugal, with the rest distributed in australia, belgium, canada, france, germany, italy, the netherlands, sweden and the united states.

Monkeypox virus belongs to the family of poxviridae, the genus orthopoxvirus. Poxviruses are the most structurally complex group of all animal viruses, and are the most diverse and structurally complex DNA viruses. Poxviruses are serologically and formally divided into six subgenera, with orthopoxvirus being the first subgenera of poxviruses. In the orthopoxvirus family, only four viruses, namely variola virus, vaccinia virus and monkeypox virus, can cause human infection, and all contain soluble antigens, nucleoprotein antigens and hemagglutinin, and the antigens have basically the same properties and are mutually crossed and immunized.

The form of the monkeypox virus is consistent with that of the orthopoxvirus, the appearance is round brick shape or oval shape, and the size is 200 to 300 nl. The periphery is a30 nm outer membrane, surrounding a homogeneous core body. The monkeypox virus genome is double-stranded DNA, approximately 197kb in length, and contains a 6379bp inverted terminal repeat at the end of the genome in the same but opposite orientation. The virus contains 190 open reading frames (60 amino acids), 4 of which are located in the inverted terminal repeat. The GC content of the genome is very low, about 33%, and the genome can encode various enzymes required for virus replication, so that the virus can replicate independently.

Monkeypox caused by monkeypox virus is a rare co-disease of humans and animals. The monkey pox epidemic in humans is characterized by several features, mainly occurring in africa, mostly in tropical rain forests, and in rare inhabitants. The rainfall is large and the outdoor activities of the crowd are increased due to the fact that the rainfall is large in 6-8 months. Furthermore, one of the most important features of the monkeypox epidemic is that transmission between humans and humans is very rare.

The disease is primarily transmitted through animals, and humans can infect monkeypox because of bites in infected animals or direct contact with blood, body fluids and rashes of infected animals. In addition, the virus can be transmitted from person to person, mainly through respiratory droplets in long-term, direct face-to-face contact; additionally, monkeypox can also be transmitted by direct contact with the body fluids or contaminated articles of a patient suffering from monkeypox. All age groups may be infected with the disease, but the risk of death in unsupervised children is high, with a fatality rate of approximately 10%.

The current laboratory diagnosis method of the monkeypox virus is divided into three categories, the first category is a culture and separation method, and the monkeypox virus is separated out through cell culture; the second type is electron microscope inspection, which detects whether the virus with the same form as the orthopoxvirus exists in the sample to be detected; the third is a molecular diagnosis method, which adopts a fluorescence PCR method to specifically detect the monkeypox virus nucleic acid in a sample to be detected. However, although the culture separation method has accurate results, the detection period is too long, false negative results are easy to occur, and the requirements on the biological safety of a laboratory are extremely high due to the live virus operation; the electron microscope inspection method has low sensitivity, long sample preparation complexity period, expensive and complicated electron microscope operation, and is not suitable for large-scale popularization; the molecular diagnosis method has high sensitivity and strong specificity, and has great advantages of determining whether the virus exists by adopting a molecular detection method. However, nucleic acid products for detecting monkeypox virus are still relatively few in the market at present, and in view of some molecular differences between monkeypox virus strains currently transmitted in some countries and monkeypox virus strains mainly transmitted in africa before, there is a possibility that detection omission occurs when only developing molecular detection products based on monkeypox virus strains transmitted in africa before. In addition, some of the existing molecular products may cross-react due to the high affinity of different members of the orthopoxvirus genus.

Disclosure of Invention

The invention provides a method, oligonucleotide and a kit for detecting monkeypox virus, wherein a conservative DNA fragment (such as a conservative region in an F3L gene) in a monkeypox virus genome is selected to design a first primer pair and a first probe, and a second primer pair and a second probe aiming at a human body reference gene (such as human RNase P) are introduced simultaneously, so that false negative encountered in the process of detecting a clinical sample can be effectively avoided. The detection system aiming at the monkeypox virus can effectively avoid the problem of missed detection of the monkeypox virus.

In the invention, x1 and x2 at the 5 'end of each probe are fluorescent reporter groups, and y1 and y2 at the 3' end of each probe are quenching groups which emit fluorescence or do not emit fluorescence. Preferably, the 3' end of the first probe further comprises an MGB molecule. In the present invention, the fluorescent reporter gene may be selected from one of FAM, VIC, HEX, ROX, cy5, cy5.5, etc., and the quencher group may be selected from one of TAMRA, BHQ (BHQ 1, BHQ2, or BHQ 3), QSY, NFQ, etc.

The fluorescent reporter and quencher of the first and second probes of the present invention are given in the form of, for example, "FAM-TARMA/BHQ" or "VIC/HEX/JOE-TAMRA/BHQ" - ", the left of which represents the fluorescent reporter and the right of which represents the quencher. FAM-TARMA/BHQ indicates that the fluorescence reporter of the same probe is FAM, and the quenching group can be selected from TARMA or BHQ. VIC/HEX/JOE-TAMRA/BHQ indicates that the fluorescent reporter gene of the same probe can be selected from VIC, HEX or JOE, and the quenching group can be selected from TAMRA or BHQ. FAM-MGBNFQ means that the fluorescent reporter gene of the same probe is selected from FAM and the quencher group is selected from NFQ, and meanwhile, the 3' end of the probe also contains MGB molecules. VIC/HEX/JOE-MGBNFQ represents that the fluorescent reporter gene of the same probe is selected from one of VIC, HEX and JOE, the quenching group is selected from NFQ, and meanwhile, the 3' end of the probe also contains MGB molecules.

The invention provides a conservative DNA fragment in a monkeypox virus genome, and the sequence of the conservative DNA fragment is SEQ ID NO. 7 or a complementary sequence thereof.

The invention also provides an oligonucleotide for detecting the monkeypox virus, which comprises a first primer pair and a first probe for detecting the conservative DNA segment in the monkeypox virus genome. Preferably, said conserved DNA segment is monkeypox virus specific, i.e. the DNA segment is specific for monkeypox virus.

Further, the oligonucleotide further comprises a second primer pair and a second probe for detecting the internal reference gene.

Further, the conserved DNA fragment is derived from a simian pox virus gene J1L, J2L, J3L, D1L, D2L, D3R, D4L, D5R, D6L, D7L, D8L, D9L, D10L, D11L, D12L, D13L, D14L, D15L, D16L, D17L, D18L, D19L, P1L, P2L, O1L, O2L, C1L, C2L, C3L, C4L, C5L, C6R, C7L, C8L, C9L, C10L, C11L, C12L, C13L, C14L, C15L, C16L, C17L, C18L, C19L, C20L, C21L, C22L, C23R, F1L, F2L, F3L, F4L, F5R, F6R, F7R, F8L, F9R, F10L, Q1L, Q2L, I1L, I2L, I3L, I4L, I5L, I6L, I7L, I8R, G1L, G2L, G3R, G4L, G5R, G6R, G7R, G8L, G9R, G10R, M1R, M2R, M3L, M4R, M5R, L1R, L2R, L3R, L4R, L5L, L6R, H1L, H2R, H3L, H4L, H5R, H6R, H7R, E1R, E2L, E3R, E4R, E5R, E6R, E7R, E8L, E9R, E10R, E11L, E12L, E13L, A1L, A2L, A3L, A4L, A5L, A6R, A7L, A8L, A9R, A10L, A11L, A12R, A13L, A14L, A15L, A16L, A17L, A18L, A19R, A20L, A21L, A22R, A23R, A24R, A25R, A26L, A27L, A28L, A29L, A30L, A31L, A32L, A33R, A34L, A35R, A36R, specific conserved DNA fragments among A37R, A38R, A39R, A40L, A41L, A42R, A43R, A44R, A45L, A46R, A47R, A48R, A49R, A50R, A51R, B1R, B2R, B3R, B4R, B5R, B6R, B7R, B8R, B9R, B10R, B11R, B12R, B13R, B14R, B15L, B16R, B17R, B18R, B19R, B20R, B21R, K1R, R1R, N1R, N2R, N3R, N4R, J1R, J2R, and J3R. Based on these specific conserved DNA fragments, primers and probes can be designed for each gene.

Further, the conservative DNA fragment is from the F3L gene.

Further, the sequence of the conservative DNA fragment is SEQ ID NO. 7 or a complementary sequence thereof.

Further, the base sequences of the first primer pair and the first probe are respectively SEQ ID NO. 1-3.

Further, the base sequences of the second primer pair and the second probe are respectively SEQ ID NO. 4-6.

Further, the fluorescent reporter group and the quenching group of the first probe are selected from any one of FAM-TARMA/BHQ, VIC/HEX/JOE-TAMRA/BHQ, FAM-MGBNFQ and VIC/HEX/JOE-MGBNFQ, and the fluorescent reporter group and the quenching group of the second probe are selected from any one of FAM-TARMA/BHQ, VIC/HEX/JOE-TAMRA/BHQ.

The invention also provides a method for detecting monkeypox virus, which comprises the following steps: (1) extracting nucleic acid in a sample; (2) Determining whether the monkeypox virus exists in a sample by utilizing a group of primers and probes, wherein the group of primers and probes comprise a first primer pair and a first probe, and the base sequence of the primers and probes is SEQ ID NO. 1-3.

Furthermore, the group of primers and probes also comprises a second primer pair and a second probe, and the base sequences of the primers and the probes are SEQ ID NO. 4-6.

The invention also provides a kit for detecting the monkeypox virus, which comprises fluorescent PCR reaction liquid, wherein the fluorescent PCR reaction liquid comprises oligonucleotide, and the oligonucleotide at least comprises a first primer pair and a first probe for detecting the conservative DNA segment in the monkeypox virus genome.

Further, the conservative DNA fragment is from F3L gene.

Further, the base sequences of the first primer pair and the first probe are SEQ ID NOS: 1 to 3, respectively.

Further, the fluorescent reporter group and the quenching group of the first probe are selected from any one of FAM-TARMA/BHQ, VIC/HEX/JOE-TAMRA/BHQ, FAM-MGBNFQ and VIC/HEX/JOE-MGBNFQ, and the fluorescent reporter group and the quenching group of the second probe are selected from any one of FAM-TARMA/BHQ and VIC/HEX/JOE-TAMRA/BHQ.

Further, the concentration of each primer and probe ranged from 75 to 300nM.

Further, the fluorescent PCR reaction solution further comprises 10 to 50mM Tris (pH 8.0 to 9.2), 10 to 50mM KCl, 10 to 20mM ammonium sulfate, 0.01 to 0.1% Tween 20, 0.2 to 2mg/mL BSA, 0.1 to 0.3mM dATP, 0.1 to 0.3mM dTTP, 0.1 to 0.3mM dCTP, 0.1 to 0.3mM dGTP, 0.1 to 0.3mM dUTP, and 3 to 6mM magnesium chloride.

The kit also comprises enzyme mixed liquor, wherein the enzyme mixed liquor comprises 1.5-3U Taq DNA polymerase, 0.5-3U Taq antibody and 0.1-2U TS-UNG enzyme.

Further, the kit also comprises a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent comprises a nucleic acid extracting solution 1, a nucleic acid extracting solution 2 and a nucleic acid extracting solution 3, the nucleic acid extracting solution 1 comprises 10-1000 mM Tris-EDTA, 5-8M guanidine hydrochloride, 1% -10% TritonX-100, pH 4-7, paramagnetic silicon oxide nano magnetic beads with a superparamagnetic core and a silicon oxide shell and a diameter of 80-800 nm, the nucleic acid extracting solution 2 comprises 10-1000 mM Tris-EDTA and 40% -60% absolute ethyl alcohol, and the nucleic acid extracting solution 3 comprises 10mM Tris-EDTA, pH 8-9.

Further, the kit further comprises a positive quality control which is monkey pox virus pseudovirus and a negative quality control which is 0.9% NaCl solution.

Further, the kit operation process comprises the following steps: (1) extracting sample nucleic acid by a three-step method: firstly, taking 0.5mL of sample and 0.5mL of nucleic acid extracting solution 1, standing for 10min, adsorbing magnetic beads by a magnetic frame, and then discarding supernatant; secondly, adding 400 mu L of nucleic acid extracting solution 2, uniformly mixing by vortex, standing for 5min, adsorbing magnetic beads by a magnetic frame, and then absorbing the supernatant; thirdly, adding 100 mu L of nucleic acid extracting solution 3, uniformly mixing by vortex, standing for 5min, adsorbing magnetic beads by a magnetic frame, and taking supernatant nucleic acid solution for later use; (2) preparing a fluorescent PCR reagent: uniformly mixing the fluorescent PCR reaction solution and the enzyme mixed solution, and respectively adding the sample nucleic acid extracted in the step (1) and negative quality control and positive quality control; (3) fluorescent PCR detection: and (3) performing fluorescence PCR amplification, and determining whether the monkeypox virus exists in the sample after the amplification is finished.

Has the beneficial effects that: (1) According to the invention, a conservative DNA fragment (F3L gene) in a monkey pox virus genome is selected to design a primer and a probe, and a human genome housekeeping gene RNaseP is introduced as an internal reference gene, so that result abnormity caused by a series of factors such as clinical sample sampling errors and nucleic acid extraction errors can be effectively avoided; (2) The kit can detect the monkeypox virus from a complex sample, has the advantages of high sensitivity, good specificity, quick and objective detection result and the like, and provides a reliable result for diagnosing the infection of the monkeypox virus.

Drawings

FIG. 1: the instrument used as a control detects the nucleic acid extracted by the paramagnetic particle method.

FIG. 2 is a schematic diagram: the invention relates to a detection result of nucleic acid extracted by a manual magnetic bead method.

FIG. 3: the invention provides a detection result of the positive reference substance P1.

FIG. 4: the invention provides a detection result of the positive reference substance P2.

FIG. 5 is a schematic view of: the invention provides a detection result of the positive reference substance P3.

FIG. 6: the invention provides a detection result of the positive reference substance P4.

FIG. 7 is a schematic view of: the invention provides a detection result of negative reference substances N1-N10.

FIG. 8: at 500copies/mL, the present invention is directed to F3L gene detection sensitivity verification result chart.

FIG. 9: at 250copies/mL, the present invention is directed to F3L gene detection sensitivity verification result chart.

FIG. 10:125copies/mL, the present invention is directed to F3L gene detection sensitivity verification result chart.

Detailed Description

In the method for detecting the monkeypox virus, a conserved sequence in the monkeypox virus is selected to design a primer and a probe, and a human genome housekeeping gene RNaseP is introduced as an internal reference gene (also called an internal reference gene) at the same time, so that the result abnormity caused by a series of factors such as clinical sample sampling errors, nucleic acid extraction errors and the like can be effectively avoided, and more importantly, the problem of missed detection of the monkeypox virus can be solved.

Through large-scale genome sequence comparison aiming at the prior monkey pox virus strains, monkey pox virus strains which appear in a plurality of countries in the world in 2022 and other virus members of poxviridae, the invention discovers that a particularly conservative DNA segment in the monkey pox virus genome can be used as a specific detection target of the monkey pox virus, and the sequence of the DNA segment is SEQ ID NO. 7 or a complementary sequence thereof. The primers and probes are designed according to the specially conserved DNA fragment, so that the specificity of detecting the monkeypox virus can be effectively improved, and the cross reaction is reduced. Meanwhile, the phenomenon of missed detection and error detection which can occur when primers and probes are designed by selecting genomic DNA fragments which are not conservative can be avoided.

Based on this, the conserved DNA fragment of F3L gene (as a specific detection target for detecting monkeypox virus), and the sequences of the designed primers and probes are shown in Table 1.

TABLE 1 specific sequences of primers, probes and conserved DNA fragments

Table 1 the first probe and the second probe are both TaqMan probes, each of which contains a fluorescent reporter group and a quencher group, the fluorescent reporter group can be selected from one of FAM, VIC, HEX, ROX, cy5, cy5.5, etc., and the quencher group can be selected from one of TAMRA, BHQ, QSY, NFQ, etc. Preferred fluorescent reporters are FAM and VIC because these two fluorescent reporters have the advantages of low background fluorescence and high fluorescence detection signal in fluorescent PCR detection. The quencher is selected based on the principle that the fluorescence absorption spectrum of the quencher overlaps with the emission spectrum of the fluorescent reporter. The preferred combination of fluorescent reporter and quencher for these two probes in Table 1 is FAM-MGBNFQ, VIC/HEX/JOE-BHQ. Preferably, the fluorescent reporter group is different for each probe, so that two-channel detection can be performed simultaneously in a single tube. Preferably, the 3' end of the first probe further comprises an MGB molecule.

Preferably, the fluorescent reporter group and the quencher group of the first probe are selected from any one of FAM-TARMA/BHQ, VIC/HEX/JOE-TAMRA/BHQ, FAM-MGBNFQ and VIC/HEX/JOE-MGBNFQ, and the fluorescent reporter group and the quencher group of the second probe are selected from any one of FAM-TARMA/BHQ and VIC/HEX/JOE-TAMRA/BHQ.

Before performing fluorescent PCR, nucleic acid (RNA) in a clinical sample needs to be extracted, and various methods such as an alkaline lysis method, a magnetic bead method and a column extraction method are used for extracting the nucleic acid. For convenience of illustration, the nucleic acid extraction reagent used in the invention adopts a magnetic bead method to extract nucleic acid in a clinical sample, and comprises a nucleic acid extracting solution 1, a nucleic acid extracting solution 2 and a nucleic acid extracting solution 3, wherein the nucleic acid extracting solution 1 comprises 10-1000 mM Tris-EDTA, 5-8M guanidine hydrochloride and 1% -10% TritonX-100, the pH value is 4-7, a superparamagnetic silica nano magnetic bead (the diameter of the magnetic bead is between 80-800 nm, and the magnetic bead has a core-shell structure, namely a superparamagnetic core and a silica shell), the nucleic acid extracting solution 2 comprises 10-1000 mM Tris-EDTA,40% -60% absolute ethyl alcohol, and the nucleic acid extracting solution 3 comprises 10mM Tris-EDTA, the pH value is 8-9.

However, it should be noted that although the manual magnetic bead method is used to extract nucleic acid from clinical samples, the present invention is not limited to this nucleic acid extraction method, and methods such as instrumental magnetic bead method and column extraction method can be used to extract nucleic acid from clinical samples.

For convenience of explanation, in the following examples, FAM-MGBNFQ was used as a fluorescent reporter group and a quencher group of a probe for detecting the F3L gene of monkeypox virus, and VIC-BHQ1 was used as a fluorescent reporter group and a quencher group of a probe for detecting the human genome housekeeping gene RNaseP. The positive quality control used in the examples of the present invention was monkeypox virus pseudovirus.

The following examples further illustrate the invention. These examples are not intended to limit the scope of the invention but rather to provide a further understanding of the invention.

Example 1: extraction of nucleic acid from clinical specimens

For convenience of explanation, in this embodiment, a normal human oropharyngeal swab and serum are used as clinical samples, nucleic acid extraction of the clinical samples is performed by the manual magnetic bead method described in the present invention, and an extraction kit of the instrumental magnetic bead method is used as a control for comparison.

10 samples of normal human oropharyngeal swabs and serum were taken, and sample DNA was extracted according to the three-step method of the present invention: taking 0.5mL of sample and 0.5mL of nucleic acid extracting solution 1, standing for 10min, adsorbing magnetic beads by a magnetic frame, and then discarding the supernatant; secondly, adding 400 mu L of nucleic acid extracting solution 2, uniformly mixing by vortex, standing for 5min, adsorbing magnetic beads by a magnetic frame, and then absorbing the supernatant; and thirdly, adding 100 mu L of nucleic acid extracting solution 3, uniformly mixing by vortex, standing for 5min, adsorbing magnetic beads by a magnetic frame, and taking supernatant nucleic acid solution for later use.

The nucleic acid extraction method is a manual magnetic bead method, and a three-step method is adopted to respectively perform cracking, washing and elution on clinical samples. Nucleic acid extraction kit (magnetic bead method) produced by Aikang Biotechnology (Hangzhou) Co., ltd.) as a control was manufactured by Aikang Biotechnology (Hangzhou) Co., ltd

The same clinical samples are subjected to nucleic acid extraction (namely an instrument magnetic bead method) on a nucleic acid purifier, the kit provided by the invention is used for detecting human genome housekeeping gene RNaseP, the detected Ct values are compared, the comparison result is shown in table 2, and the amplification result is shown in fig. 1 and fig. 2. As is clear from FIGS. 1 and 2, the nucleic acid isolation method (manual magnetic bead method) of the present invention substantially matches the detection results of nucleic acid isolation by the instrumental magnetic bead method.

TABLE 2 comparison of the results of nucleic acid extraction

Example 2: detection of monkeypox virus by using fluorescent PCR method

In the embodiment, the kit is used for carrying out fluorescence PCR detection on the monkeypox virus, and comprises fluorescence PCR reaction liquid, enzyme mixed liquid, positive quality control and negative quality control. Preferably, the kit may further comprise the nucleic acid extracting solution 1, the nucleic acid extracting solution 2 and the nucleic acid extracting solution 3 in example 1. The fluorescent PCR reaction solution preparation system is shown in Table 3. The enzyme mixture preparation system is shown in Table 4. The positive quality control was monkey pox virus pseudovirus and the negative quality control was 0.9% NaCl solution.

TABLE 3 fluorescent PCR reaction solution preparation System TABLE (1 part)

TABLE 4 enzyme mixture preparation system (1 part)

When the pathogen monkeypox virus in a clinical sample is detected, the fluorescent PCR reaction solution and the enzyme mixed solution are uniformly mixed, and the required sample number is calculated by n parts [ n = clinical sample number +1 tube positive control +1 tube negative control ]. mu.L of the fluorescent PCR reaction solution and 2. Mu.L of the enzyme mixture were added to different reaction tubes, and then 5. Mu.L of the nucleic acid solution extracted in example 1, 5. Mu.L of the negative control, and 5. Mu.L of the positive control were added to the different reaction tubes, respectively.

The reaction tubes were placed in a fluorescence PCR apparatus (ABI Q5) in a certain order, and fluorescence PCR amplification was carried out according to the following procedure, the amplification procedure being shown in Table 5:

TABLE 5 fluorescent PCR amplification procedure

After the fluorescent PCR amplification is finished, whether the monkeypox virus exists or not is detected according to Ct values of an FAM channel and a VIC channel, and the detection result is judged as shown in table 6.

TABLE 6 test results Cutoff values and results interpretation

a. Monkey pox virus gene detection interpretation standard:

1) Negative: ct value was not detected;

2) Positive: the amplification curve is S-shaped, and the Ct value is less than or equal to 40;

3) And (3) suspicious: the amplification curve is S-shaped, and the Ct value is more than 40 and less than or equal to 45, and the reinspection is needed; if the rechecking results are consistent, the result is judged to be positive.

b. Monkey pox virus nucleic acid detection positive interpretation standard:

if the detection result of the target gene is positive, the monkey pox virus nucleic acid is judged to be positive.

And c, if the reference gene VIC channel of RNaseP is negative, indicating that the sample contains PCR reaction inhibitor, extraction fails or sample is leaked in the experimental process, and suggesting re-sampling, extraction and testing. The detection result of the monkeypox virus nucleic acid is positive in a few samples possibly caused by the sampling problem, the internal reference has no amplification signal or the Ct value is more than 40, the positive result is still credible in the situation, and the sampling and retesting are carried out to confirm if necessary.

The common test results of clinical samples are shown in Table 7, and other abnormal results need to be interpreted in combination with clinical results.

TABLE 7 interpretation of common results in clinical samples

Example 3: clinical sample validation

The clinical study of the monkeypox virus real-time PCR detection kit was performed using mock clinical samples, including lesion exudate swab, lesion crust, serum or oropharyngeal swab, each sample type comprising 10 negative samples, 10 mock specimens (monkeypox virus pseudoviruses), 40 positive specimens and 40 negative specimens. The samples were validated with a control Kit (monkypox Virus Real Time PCR Kit (Shanghai ZJ Bio-Tech co., ltd.)) and the results compared.

The analysis of the detection results of the present invention for the F3L gene of monkeypox virus is shown in Table 8.

TABLE 8 nucleic acid assay clinical validation results for monkeypox virus

Positive coincidence rate = [ 40/(0 + 40) ] × 100% =100%

Negative coincidence rate = [ 40/(0 + 40) ] × 100% =100%

Total coincidence rate = [ (40 + 40)/(40 + 40) ] × 100% =100%

Relative sensitivity =100% (95% confidence interval: 91.19% -100.00%)

Relative specificity =100% (95% confidence interval: 91.19% -100%)

Accuracy =100% (95% confidence interval: 91.19% -100.00%)

Kappa＝1

The Kappa analysis criteria are as follows:

Kappa	standard of merit
		＝1	Indicate complete agreement
＞0.75	Shows good consistency
		＜0.4	Indicating poor consistency
＝0	Indicating that two results are due to chance

Clinical tests show that the Kappa value of the detection result of the reagent and the control result in the consistency analysis is 1, and the correlation between the detection result of the reagent and the control result is good.

Example 4 reference System establishment and evaluation

In this example, the following reference materials were set for the kit of the present invention to evaluate the performance.

Setting a positive reference product: selecting pseudovirion containing a fragment of the DNA of a monkeypox virus at a concentration of 1X 10 ⁴ copies/mL～1×10 ⁶ 4 positive reference samples in the range of copies/mL, labeled P1-P4.

Setting a negative reference product: selecting 2 parts of vaccinia virus nucleic acid extract, labeled N1-N2;1 part of vaccine virus nucleic acid extract, marked as N3;1 monkey pox virus negative sample nucleic acid extract, marked as N4;

the reference substance set in the kit was evaluated for the rate of coincidence of yin and yang according to the method described in example 2. The results are shown below:

the detection results of the positive reference products P1-P4 are positive FAM and VIC channel detection results. As shown in fig. 3-6.

The detection results of the negative reference products N1-N4 are that the FAM channel detection results are negative, and the VIC channel detection results are positive. As shown in fig. 7.

Example 5 assay sensitivity

The pseudovirions containing the monkeypox virus DNA fragment were tested 20 times at different concentrations (S1: 500copies/mL, S2:250copies/mL, S3:125copies/mL, respectively) according to the method described in example 2, and the lower detection limit of the kit was confirmed at a detection rate of 95%. The detection results are shown in tables 9-11 in detail, the detection rates of the F3L gene amplification primer probes for 500 and 250copies/mL pseudovirions are both 100%, and the detection rate of the F3L gene amplification primer probe for 125copies/mL pseudovirions is 75%. In conclusion, the lower detection limit of the kit is 250copies/mL. As shown in fig. 8-10.

TABLE 9 statistics of the test results of the test Limit samples S1

Detection limit sample S1	F3L gene
		1	37.04
2	37.11
		3	36.93
4	36.76
		5	36.33
6	37.00
		7	36.23
8	35.94
		9	36.05
10	35.96
		11	38.34
12	36.54
		13	36.23
14	36.70
		15	35.98
16	37.43
		17	36.28
18	37.43
		19	35.87
20	36.93
		Percentage of detection (%)	100％

TABLE 10 statistics of detection limit sample S2

TABLE 11 statistics of detection Limit samples S3

Example 6 reaction specificity analysis

In this example, cross-over samples of other coronaviruses, respiratory pathogens, and the like were validated against the kit of the present invention. The results are shown in table 13, and it can be seen from the results that the kit of the present invention does not have cross reaction to the detection of other coronaviruses and respiratory pathogens, and thus the kit of the present invention has good specificity.

TABLE 12 Cross-pathogen validation results

Sequence listing

<110> Aikang Biotechnology (Hangzhou) Co., ltd

Specific detection target of <120> monkeypox virus, oligonucleotide and kit thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tacatcatct attatagcat cagcatca 28

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atactcctcc tcgttggtct agga 24

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgctgatac acggccta 18

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctaagcacac gcctattaat ttagtgc 27

<210> 5

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agcaagtaaa gtttgaaacc tagtgatg 28

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tggcaaatct accaatggtt ctaaagccga 30

<210> 7

<211> 113

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atcctctctc attgattttt cgcgggatac atcatctatt atagcatcag catcagaatc 60

tgtaggccgt gtatcagcat ccattgtcgt agaccaacga ggaggagtat cgt 113

Claims

1. A monkey pox virus specificity detection target, characterized in that, the sequence of the detection target is SEQ ID NO 7 or its complementary sequence.

2. Oligonucleotide for the detection of monkeypox virus, characterized in that it comprises at least: a first primer pair and a first probe for detecting the simian pox virus genome conservative DNA segment.

3. The oligonucleotide of claim 2, further comprising a second primer pair and a second probe for detecting an internal reference gene.

4. The oligonucleotide of claim 2, wherein the conserved DNA segment is from the F3L gene.

5. The oligonucleotide of claim 2, wherein the sequence of the conserved DNA segment is SEQ ID NO 7 or its complement.

6. The oligonucleotide of claim 2, wherein the base sequences of the first primer pair and the first probe are SEQ ID NOS: 1 to 3, respectively.

7. The oligonucleotide of claim 3, wherein the base sequences of the second primer pair and the second probe are SEQ ID NOS.4 to 6, respectively.

8. The oligonucleotide of claim 3, wherein the fluorescent reporter and quencher of the first probe are selected from the group consisting of FAM-TARMA/BHQ, VIC/HEX/JOE-TAMRA/BHQ, FAM-MGBNFQ and VIC/HEX/JOE-MGBNFQ, and the fluorescent reporter and quencher of the second probe are selected from the group consisting of FAM-TARMA/BHQ, VIC/HEX/JOE-TAMRA/BHQ.

9. A kit for detecting monkeypox virus, said kit comprising a fluorescent PCR reaction solution, characterized in that said fluorescent PCR reaction solution comprises an oligonucleotide according to any one of claims 2 to 8.

10. The kit according to claim 9, further comprising nucleic acid extraction reagents comprising a nucleic acid extract 1, a nucleic acid extract 2 and a nucleic acid extract 3, wherein the nucleic acid extract 1 comprises 10-1000 mM Tris-EDTA, 5-8M guanidine hydrochloride and 1-10% triton x-100, pH 4-7, paramagnetic silica nanobead, the nucleic acid extract 2 comprises 10-1000 mM Tris-EDTA, 40-60% absolute ethanol, and the nucleic acid extract 3 comprises 10mM Tris-EDTA pH 8-9.