A monkeypox virus fluorescence PCR detection kit and method

Abstract

The present invention proposes a monkeypox virus fluorescent PCR detection kit and method, wherein the kit includes a premixed reaction solution, a positive control and a negative control, the premixed reaction solution includes a gene multiple reaction solution, an enzyme mixture and a nucleic acid amplification reaction solution, the gene multiple reaction solution includes a monkeypox virus F3L gene upstream primer, a downstream primer and a probe, whose sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and an internal standard GAPDH gene upstream primer, downstream primer and probe, whose sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The kit of the present invention is a premixed reaction solution, which avoids the error caused by mixing the reaction solution and enzyme, reduces the difficulty of operation, and greatly improves the detection sensitivity; and can effectively detect the Congo Basin branch and the West African branch at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of PCR detection, and in particular to a monkeypox virus fluorescence PCR detection kit and method. Background Art

[0002] Monkeypox virus (MPXV) belongs to the Poxviridae family, the Chorpopoxvirinae subfamily, and the Orthopoxvirus genus. It is a zoonotic double-stranded DNA virus approximately 197 kb in length. Its genome contains closed hairpins at both ends and inverted terminal repeats. The virion is composed of over 90% protein, containing over 100 polypeptides and more than 10 enzymes. Other members of the Orthopoxvirus family include smallpox virus, vaccinia virus, cowpox virus, mousepox virus, and raccoonpox virus. Monkeypox and the first three of these viruses can infect humans. The morphology, chemical structure, antigenicity, pathogenicity, and resistance of MPXV are similar to those of other poxviruses, requiring differentiation from smallpox and varicella. Based on the clinical manifestations and epidemiological characteristics of MPXV infection, combined with genotypic studies, MPXV is divided into the Congo Basin clade (CB) and the West African clade (WA).

[0003] my country's "Monkeypox Diagnosis and Treatment Guidelines (2022 Edition)" categorizes monkeypox diagnosis into suspected and confirmed cases. Diagnosis is based on epidemiological history, clinical manifestations, and etiological testing, with a positive monkeypox virus nucleic acid test serving as a key indicator of etiological diagnosis for confirmed cases. Real-time fluorescence PCR using clinical specimens has a short window period, high sensitivity, and good specificity. It can detect monkeypox genome fragments in skin lesion specimens from monkeypox patients, providing a basis for rapid laboratory diagnosis in the clinic.

[0004] CN114752711A discloses a kit for detecting monkeypox virus CB and monkeypox virus WA. However, the kit has a low sensitivity (200 copies / mL), and the fluorescent PCR reaction solution and enzyme mixture are separate. Other components of the kit, the reaction system, and the amount of sample added all affect the sensitivity, specificity, and reproducibility of the assay. Therefore, there is a need for a kit with high sensitivity and specificity that can effectively detect both the Congo Basin clade and the West African clade. Summary of the Invention

[0005] In view of this, the present invention proposes a kit with good detection sensitivity and specificity and capable of effectively detecting the Congo Basin branch and the West African branch at the same time.

[0006] The technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a monkeypox virus fluorescent PCR detection kit, comprising a premixed reaction solution, a positive control, and a negative control, wherein the premixed reaction solution comprises a gene multiplex reaction solution, an enzyme mixture, and a nucleic acid amplification reaction solution, wherein the gene multiplex reaction solution comprises an upstream primer, a downstream primer, and a probe for the monkeypox virus F3L gene, whose sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and an upstream primer, a downstream primer, and a probe for the internal standard GAPDH gene, whose sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0007] Based on the above technical solution, preferably, the upstream primer concentration of the monkeypox virus F3L gene is 0.3-0.5 μmol / L, the downstream primer concentration is 0.3-0.5umol / L, and the probe concentration is 0.1-0.3umol / L; the upstream primer concentration of the internal standard GAPDH gene is 0.1-0.3umol / L, the downstream primer concentration of the internal standard GAPDH gene is 0.1-0.3umol / L, and the internal standard GAPDH gene probe concentration is 0.1-0.3umol / L.

[0008] Based on the above technical solution, preferably, the probes shown in SEQ ID NO: 3 and SEQ ID NO: 6 are both labeled with a fluorescent group at their 5' ends, and are both labeled with a quenching group and a PO4 group at their 3' ends.

[0009] On the basis of the above technical solution, preferably, the fluorescent group is one or more of HEX, JOE, VIC and ROX, and the quenching group is one or more of TAMRA, BHQ1, BHQ2 and BHQ3.

[0010] Based on the above technical solution, preferably, the fluorescent groups labeled with the probes shown in SEQ ID NO: 3 and SEQ ID NO: 6 are different.

[0011] Based on the above technical solution, preferably, the enzyme mixture includes Taq polymerase and UNG enzyme.

[0012] Based on the above technical solution, preferably, the nucleic acid amplification reaction solution includes MgSO4, Buffer, dNTPs and an enhancer.

[0013] Based on the above technical solution, preferably, the enhancer includes glycerol, BSA and TMAC.

[0014] Based on the above technical solution, preferably, the positive control is virus-like particles of the monkeypox virus F3L gene fragment, GAPDH plasmid particles and 50% TE Buffer, and the negative control is 0.9% sodium chloride solution.

[0015] In a second aspect, the present invention provides a fluorescent PCR detection method for detecting monkeypox virus for non-diagnostic purposes, comprising the following steps:

[0016] S1, extracting or releasing nucleic acid from the sample to be tested;

[0017] S2, performing fluorescent PCR analysis on the nucleic acid obtained in S1 using the above kit;

[0018] S3, obtain and analyze the results.

[0019] The present invention's fluorescent PCR detection kit for monkeypox virus and its method of use offer the following advantages over existing technologies: The kit utilizes a premixed reaction solution, eliminating errors associated with mixing the reaction solution and enzyme, reducing operational complexity and significantly improving detection sensitivity. The kit incorporates locked nucleic acid (LNA) probes, which exhibit high recognition and affinity, significantly increasing the Tm difference between perfectly matched and mismatched bases. This allows for more effective resolution of differences down to a single base, thereby significantly improving the detection rate of monkeypox gene mutations. The kit also exhibits excellent specificity, enabling effective detection of both the Congo Basin and West African clades. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a fluorescence amplification curve diagram of the monkeypox F3L gene and the internal reference GAPDH gene of the present invention;

[0022] Figure 2 This is a sensitivity verification result diagram of the kit of the present invention;

[0023] Figure 3 is a fluorescence amplification curve diagram of the monkeypox channel of the present invention;

[0024] Figure 4 It is a specific detection diagram of the kit of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1

[0027] The present invention provides a monkeypox virus fluorescent PCR detection kit, comprising a premixed reaction solution, a positive control, and a negative control. The premixed reaction solution comprises an F3L / GAPDH gene multiplex reaction solution, an enzyme mixture, and a nucleic acid amplification reaction solution. The gene multiplex reaction solution comprises an upstream primer, a downstream primer, and a probe for the monkeypox virus F3L gene, whose sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and an upstream primer, a downstream primer, and a probe for the internal standard GAPDH gene, whose sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0028] The probes shown in SEQ ID NO: 3 and SEQ ID NO: 6 are both labeled with a fluorescent group at their 5' ends and a quenching group and a PO4 group at their 3' ends.

[0029] The fluorescent group is one or more of HEX, JOE, VIC and ROX, and the quenching group is one or more of TAMRA, BHQ1, BHQ2 and BHQ3.

[0030] The fluorescent groups labeled with the probes shown in SEQ ID NO: 3 and SEQ ID NO: 6 are different.

[0031] The primer and probe sequences for the monkeypox virus F3L gene and the internal standard GAPDH gene are shown in Table 1:

[0032] Table 1 Primers and probes

[0033]

[0034] The concentration of the upstream primer of the monkeypox virus F3L gene is 0.3-0.5 μmol / L, preferably 0.4 μmol / L; the concentration of the downstream primer is 0.3-0.5 μmol / L, preferably 0.4 μmol / L; and the probe concentration is 0.1-0.3 μmol / L, preferably 0.2 μmol / L.

[0035] The concentration of the internal reference GAPDH upstream primer is 0.1-0.3umol / L, preferably 0.2μmol / L; the concentration of the internal reference GAPDH downstream primer is 0.1-0.3umol / L, preferably 0.2μmol / L; the concentration of the internal reference GAPDH probe is 0.1-0.3umol / L, preferably 0.1μmol / L.

[0036] The enzyme mixture included 5 U / μL Taq polymerase and 2 U / μL UNG enzyme.

[0037] The nucleic acid amplification reaction solution included 2.5 mmol / L MgSO4, 5 μL 10× Buffer, 10 mmol / L dNTPs, and an enhancer, which included 10% glycerol, 50 mg / mL BSA, and 30 mmol / L TMAC.

[0038] The positive control was virus-like particles of the monkeypox virus F3L gene fragment, GAPDH plasmid particles and 50% TEBuffer, and the negative control was 0.9% sodium chloride solution.

[0039] Example 2

[0040] A fluorescent PCR detection method for detecting monkeypox virus for non-diagnostic purposes comprises the following steps:

[0041] S1, extract or release the nucleic acid of the sample to be tested, and the sample is blood.

[0042] S2, amplification, using the kit in Example 1 to perform fluorescent PCR analysis on the nucleic acid obtained in S1.

[0043] The components and contents of the PCR reaction solution in this example are shown in Table 2.

[0044] Table 2 PCR reaction solution components and concentrations

[0045]

[0046] Take pseudovirus particles containing monkeypox virus F3L gene and dilute them to 10 with negative serum. 5 copies / mL, 10 4 copies / mL, 10 3 The nucleic acid was extracted using the nucleic acid extraction reagents from Wuhan Biotech Gene Engineering Co., Ltd., of which 10 5 The samples were extracted 10 times at 100 copies / mL and 20 times at 100 copies / mL.

[0047] (2) According to the PCR amplification system in this article Figure 2Prepare the reaction system according to 45 μL per reaction, and mix 45 μL reaction solution + 5 μL sample nucleic acid per reaction.

[0048] (3) RT-PCR amplification was performed using a Shanghai Hongshi SLAN 96P fluorescence quantitative PCR instrument. The procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, collecting fluorescence signals, and performing 40 cycles of denaturation and annealing. The results are shown in Figure 1-2 .

[0049] (4) The presence of Toxoplasma gondii virus in the sample is determined based on the fluorescence signal strength and cycle threshold of the channel amplification template.

[0050] Detection channels and reference fluorescence: The fluorescence PCR instrument detection channels are: FAM and VIC, and the reference fluorescence is set to none. The genes detected by each channel are shown in the table below.

[0051] Table 3 Detection channels

[0052]

[0053] The requirements for determining the validity of the experiment are as follows: For the FAM channel, the CT value of the negative control must be ≥38 or display "Undet"; the CT value of the strong positive control should be <33.0; the CT value of the critical positive control should be greater than the CT value of the strong positive control and <38.0.

[0054] When the negative control / positive control and endogenous reference test results of each test target meet the requirements, the principles for judging the positiveness of the test sample are as follows:

[0055] If the Ct value of the FAM channel test is less than 38, the test sample is positive;

[0056] If the FAM channel CT is ≥38 and the VIC channel CT is ≤36, the sample is negative;

[0057] If the CT of the FAM channel is 38≤ Ct<40, retesting is required. It is recommended to re-extract the nucleic acid of the sample and amplify and test it simultaneously with the nucleic acid extracted from the sample previously. Combining the two tests, if the CT of the FAM channel is both <38, it can be determined that the monkeypox virus is positive. Otherwise, it should be reported as "suspicious".

[0058] Figure 3 As shown, the primer and internal standard primer channels are at 10 5 copies / mL, 10 4 copies / mL, 10 3At concentrations of 0.1 copies / mL and 100 copies / mL, there were obvious S-shaped fluorescence amplification curves, showing positive results, indicating that the detection limit of this product can reach 100 copies / mL.

[0059] The above primer-probe combination, PCR reaction solution and nucleic acid amplification detection method were used to perform repeatability testing. The kit was used to perform repeatability testing 10 times, and the test results are shown in Table 4:

[0060] Table 4 Repeatability test results

[0061]

[0062] As shown in Table 4: The concentration is 10 5 copies / mL, the CV value of 10 repeated tests was 0.09%, which was less than 5%, indicating that the kit had good reproducibility.

[0063] To demonstrate the cross-reactivity of the test kit, the above test kit and detection method were used to verify the specificity of the detection system. Smallpox, chickenpox, herpes zoster, herpes simplex, measles, cowpox, mousepox, dengue fever, scabies, and syphilis viruses were tested. The detection method is as follows:

[0064] (1) Smallpox, chickenpox, herpes zoster, herpes simplex, measles, cowpox, mousepox, dengue fever, scabies and syphilis viruses were collected and nucleic acid was extracted using the nucleic acid extraction reagent of Wuhan Baitai Gene Engineering Co., Ltd.

[0065] (2) Prepare the reaction system according to the PCR amplification system described in this article, with 45 μL per reaction.

[0066] (3) Mix 45 μL of reaction solution and 5 μL of sample nucleic acid per reaction.

[0067] (4) PCR amplification: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, collecting fluorescence signals, denaturation and annealing for 40 cycles, the results are shown in Figure 4 .

[0068] Figure 4 As far as we know, all the results showed no cross-reaction with the test kit and smallpox, chickenpox, herpes zoster, herpes simplex, measles, cowpox, mousepox, dengue fever, scabies, and syphilis virus samples, indicating good specificity.

[0069] The comparative PCR reaction solution was not premixed; the enzyme and other components were stored separately and prepared on-site. Furthermore, the probes were not modified with locked nucleic acid, and no enhancer was added to the PCR reaction system. Otherwise, the reagent concentrations and test sample concentrations in the PCR reaction system were the same as in the example.

[0070] Test results showed that the comparative kit exhibited poor reproducibility after 10 tests, with a CV value >5%. Furthermore, when the comparative kit was used to detect samples of smallpox, varicella, herpes zoster, herpes simplex, measles, cowpox, mousepox, dengue fever, scabies, and syphilis viruses, cross-reactions between the comparative kit and the varicella and herpes zoster viruses occurred, resulting in false positives and poor specificity.

[0071] The locked nucleic acid (LNA) probes of the present invention possess excellent recognition and strong affinity for both DNA and RNA, and their high nuclease resistance makes them resistant to enzymatic degradation. The complex in which the LNA probe binds to the RNA / DNA chain exhibits high thermal stability but is sensitive to base mismatches. A single base mismatch can significantly decrease the melting temperature (Tm) of the RNA / DNA chain, significantly increasing the Tm difference between perfectly matched and mismatched bases. During PCR amplification, the LNA can bind to the wild-type sequence, thereby blocking PCR extension and causing amplification failure of the wild-type gene. However, the LNA cannot bind to the mutant sequence, allowing amplification of the monkeypox F3L gene mutant, thereby significantly improving the detection rate of the monkeypox F3L gene mutation.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monkeypox virus fluorescence PCR detection kit, characterized in that: The invention comprises a premixed reaction solution, a positive control and a negative control. The premixed reaction solution comprises a gene multiplex reaction solution, an enzyme mixture and a nucleic acid amplification reaction solution. The gene multiplex reaction solution comprises an upstream primer, a downstream primer and a probe of the monkeypox virus F3L gene, whose sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and an LNA probe sequence of 5'-FAM-CTAGGCCCCACTGATTCAAt-TAMRA-PO4-3'; and an upstream primer, a downstream primer and a probe of the internal standard GAPDH gene, whose sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and an LNA probe sequence of 5'-VIC-CCACCACCCTGTTGCTGTAg-3'. The nucleic acid amplification reaction solution comprises an enhancer, and the enhancer comprises glycerol, BSA and TMAC.

2. A kit for detecting monkeypox virus by fluorescent PCR according to claim 1, characterized in that: The upstream primer concentration of the monkeypox virus F3L gene is 0.3-0.5 μmol / L, the downstream primer concentration is 0.3-0.5 μmol / L, and the probe concentration is 0.1-0.3 μmol / L; the upstream primer concentration of the internal standard GAPDH gene is 0.1-0.3 μmol / L, the downstream primer concentration of the internal standard GAPDH gene is 0.1-0.3 μmol / L, and the internal standard GAPDH gene probe concentration is 0.1-0.3 μmol / L.

3. A monkeypox virus fluorescent PCR detection kit according to claim 1, characterized in that: The probes shown in SEQ ID NO: 3 and SEQ ID NO: 6 are both labeled with a fluorescent group at their 5' ends, and with a quenching group and a PO4 group at their 3' ends.

4. A monkeypox virus fluorescent PCR detection kit according to claim 3, characterized in that: The fluorescent group is one or more of HEX, JOE, VIC and ROX, and the quenching group is one or more of TAMRA, BHQ1, BHQ2 and BHQ3.

5. A monkeypox virus fluorescent PCR detection kit according to claim 4, characterized in that: The fluorescent groups labeled with the probes shown in SEQ ID NO: 3 and SEQ ID NO: 6 are different.

6. A kit for detecting monkeypox virus by fluorescent PCR according to claim 1, characterized in that: The enzyme mixture includes Taq polymerase and UNG enzyme.

7. The kit for detecting monkeypox virus by fluorescent PCR according to claim 1, wherein: The nucleic acid amplification reaction solution further includes MgSO4, Buffer, and dNTPs.

8. The kit for detecting monkeypox virus by fluorescent PCR according to claim 1, wherein: The positive control was virus-like particles of the monkeypox virus F3L gene fragment, GAPDH plasmid particles and 50% TE Buffer, and the negative control was 0.9% sodium chloride solution.

9. A fluorescent PCR method for detecting monkeypox virus for non-diagnostic purposes, characterized in that: The steps include: S1, extracting or releasing nucleic acid from the sample to be tested; S2, performing fluorescent PCR analysis on the nucleic acid obtained in S1 using the kit according to any one of claims 1 to 8; S3, obtain and analyze the results.

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