Monkeypox and monkey B virus fluorescent PCR kit and detection method

Abstract

The application discloses a fluorescent PCR kit for simultaneously detecting monkeypox and monkey B virus, and the kit comprises a primer mixture capable of simultaneously detecting monkeypox virus and monkey B virus, wherein the primer sequence for detecting the monkeypox virus is shown as SEQ ID No. 01 and SEQ ID No. 02; and the primer sequence for detecting the monkey B virus is shown as SEQ ID No. 03 and SEQ ID No. 04. The application further provides a method for simultaneously detecting monkeypox virus and monkey B virus by using the fluorescent PCR kit, and the method comprises the following steps: 1) specifically performing PCR amplification on the monkeypox virus and the monkey B virus; and 2) generating a melting curve after the amplification is completed, so as to distinguish the monkeypox virus and the monkey B virus by using the difference between the melting curve Tm values. The application has the characteristics of being capable of rapidly and accurately detecting the monkeypox virus and the monkey B virus simultaneously.

Description

Technical Field

[0001] This invention belongs to the field of virus detection. More specifically, this invention relates to a fluorescent PCR kit and detection method for simultaneously detecting monkeypox and monkey B virus. Background Technology

[0002] Monkeypox virus and monkey B virus are both serious zoonotic viruses that pose a serious or potential zoonotic threat to primate veterinarians, animal care workers, and laboratory researchers.

[0003] Monkeypox virus is an enveloped double-stranded DNA virus that can be subdivided into two strains: the West African strain and the Congo Basin strain. The Congo strain is more pathogenic, with clinical symptoms in humans similar to smallpox and a mortality rate as high as 10%. The primary hosts of monkeypox virus are rodents, such as squirrels and Gambian rats, and it can be transmitted to monkeys and humans through direct contact with blood or bodily fluids. Current records indicate that handling infected monkeys or squirrels is the main cause of transmission in Africa. Human-to-human transmission of monkeypox virus mainly occurs through contact with contaminated objects and through airborne droplets. Currently, there is no specific vaccine for preventing monkeypox virus, but cross-protection has been confirmed in individuals vaccinated against smallpox. However, with the eradication of smallpox, vaccination has ceased, and this cross-protection is weakening. Since May 19, 2022, hundreds of cases of monkeypox have been discovered globally, and the number of confirmed cases worldwide is expected to continue to rise.

[0004] Monkeypox virus (Sinobothian b virus) is a double-stranded DNA herpesvirus A circulating in rhesus monkeys. Without treatment, the mortality rate in humans infected with Sinobothian b virus is as high as 80%. Rhesus monkeys are the natural host of Sinobothian b virus, but other primate vertebrates, including non-human primates, can also be infected. Transmission of Sinobothian b virus occurs through direct contact, whether between animals, between animals and humans, between humans, or between contaminated objects and animals or humans. Currently, there is no specific vaccine for preventing monkeypox virus; timely antiviral treatment is the most effective way to reduce the mortality rate of Sinobothian b virus.

[0005] In summary, both monkeypox and monkey B virus have high mortality rates, and no specific vaccines have yet been developed for either virus. Therefore, developing a highly sensitive and time-efficient test kit that can simultaneously detect both viruses would enhance surveillance among workers in relevant occupations, ensure timely detection and treatment of infected individuals, and eliminate the virus as early as possible in its development, thereby reducing the risk of group transmission. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] One objective of this invention is to provide a fluorescent PCR kit and detection method for the simultaneous detection of monkeypox and monkey B virus, which features rapid reaction and high sensitivity, enabling rapid and accurate simultaneous detection of monkeypox virus and monkey B virus.

[0008] To achieve these objectives and other advantages of the present invention, a fluorescent PCR kit for simultaneous detection of monkeypox virus and monkey B virus is provided. The kit comprises a primer mixture capable of simultaneously detecting monkeypox virus and monkey B virus. The primer sequences for detecting monkeypox virus are shown in SEQ ID No. 01 and SEQ ID No. 02; and the primer sequences for detecting monkeypox virus are shown in SEQ ID No. 03 and SEQ ID No. 04.

[0009] Preferably, the kit further comprises a nonspecific fluorescent dye, which is one of SYBR Green, Eva Green, Helix Green and LC Green.

[0010] Preferably, the kit further comprises a PCR buffer containing the following components: N-Taq DNA polymerase 5-10 U; Tris-HCl pH 8.2-8.425 mM-75 mM; KCl 50 mM-75 mM; MgCl2

[0011] 2.5mM-3.75mM; dATP 2.5mM-7.5mM; dTTP 2.5mM-7.5mM; dCTP 2.5mM-7.5mM; dGTP 2.5mM-7.5mM; Glycerol 2.5%-12.5%; NaN 30.2%-1%.

[0012] Preferably, the PCR buffer contains the following components: 5U N-Taq DNA polymerase; 8.335mM Tris-HCl; 60mM KCl; 3mM MgCl2; 4mM dATP; 4mM dTTP; 4mM dCTP; 4mM dGTP; 10% glycerol; and 0.5% NaN.

[0013] Preferably, the kit further includes a positive control and a negative control, wherein the positive control is prepared from a plasmid mixture of monkeypox virus and monkey B virus, and the negative control is prepared from TE buffer.

[0014] A fluorescent PCR kit for simultaneous detection of monkeypox virus and monkey B virus, comprising the following steps:

[0015] 1) Specific PCR amplification of monkeypox virus and monkeypox B virus;

[0016] 2) After amplification, a melting curve is generated to distinguish between monkeypox virus and monkey B virus by using the difference in Tm values ​​of the melting curve.

[0017] Preferably, the amplification system for amplifying monkeypox virus and monkeypox B virus is as follows: PCR buffer 10-20 μL, primers for monkeypox virus and monkeypox B virus 3-5 μL each, positive standard or negative control or sample format extraction solution 3-5 μL, and deionized water 5-10 μL.

[0018] Preferably, the primer concentration for amplifying monkeypox virus is 0.4 μM, and the primer concentration for amplifying monkey B virus is 0.35 μM.

[0019] Preferably, the amplification program for monkeypox virus and monkeypox B virus is: pre-denaturation at 5°C for 20 seconds; denaturation at 95°C for 5 seconds; annealing at 56-62°C for 20 seconds, for a total of 40-45 cycles.

[0020] Preferably, the amplification program for monkeypox virus and monkeypox B virus is as follows: pre-denaturation at 95°C for 20 seconds; denaturation at 95°C for 5 seconds; annealing at 60°C for 20 seconds, for a total of 40 cycles.

[0021] The present invention has at least the following beneficial effects:

[0022] First, the kit described in this invention is easy to operate and has a short reaction time. A single tube can identify whether monkeypox virus and monkey B virus are present in a sample within 0.5 hours.

[0023] Secondly, the kit described in this invention has high sensitivity, with a detection limit of 500 copies / ml for monkeypox virus and 500 copies / ml for monkey B virus.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0025] Figure 1 This is a curve showing the spread of monkeypox virus.

[0026] Figure 2 Standard curve for monkeypox virus;

[0027] Figure 3 This is an amplification curve of simian B virus;

[0028] Figure 4 Standard curve for simian B virus;

[0029] Figure 5 Hypoxetine curves for monkeypox and monkey B virus. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0031] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0032] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0033] I. Primer Design

[0034] First, the genome sequences of monkeypox virus and monkey B virus were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / );

[0035] Then, Clustal software was used to perform multiple sequence alignment on the downloaded monkeypox virus and monkey B virus genome sequences to find relatively conserved sequences.

[0036] Finally, specific primers were designed using Oligo software targeting conserved genome sequences of monkeypox virus and monkey B virus, respectively. The primer sequences for detecting monkeypox virus are shown in SEQ ID No. 01 and SEQ ID No. 02; and the primer sequences for detecting monkeypox virus are shown in SEQ ID No. 03 and SEQ ID No. 04.

[0037] II. PCR buffer optimization

[0038] A PCR buffer contains Taq enzyme, monovalent and divalent cations, and reaction substrates (a mixture of dNTPs) essential for PCR amplification. To enhance the specificity and sensitivity of PCR amplification, suitable PCR enhancers also need to be added to the PCR buffer. Each component in the PCR buffer only exerts its maximum effect at its optimal reaction concentration; if the concentration is too low, the effect will be insignificant, and if the concentration is too high, it may even inhibit PCR amplification. Therefore, it is necessary not only to select appropriate PCR components but also to optimize the concentrations of various components in the PCR buffer.

[0039] After optimization, the components and their concentrations in the PCR buffer of the kit of this invention are shown in Table 1:

[0040] Table 1. PCR buffer components and concentrations

[0041] Components concentration Tris-HCl at pH 8.3 35mM KCl 60mM <![CDATA[MgCl2]]> 3mM dNTPs 4mM N-Taq DNA polymerase 5U glycerin 10％ <![CDATA[NaN3]]> 0.5％

[0042] III. PCR Procedure Optimization

[0043] Traditional PCR amplification typically involves a cycle of denaturation, annealing, and extension to enrich the target fragment. This invention's kit employs a rapid PCR amplification program that combines the annealing and extension steps into one, transforming the three-step procedure into a two-step process, and reducing the reaction time for each step.

[0044] The optimized PCR amplification program of this invention is shown in Table 2:

[0045] Table 2 PCR amplification program

[0046]

[0047] IV. Standard Curve

[0048] Monkeypox and monkey B virus standards were diluted to five concentration gradients using a 10-fold serial dilution method. 7 copies / ml, 10 6 copies / ml, 10 5 copies / ml, 10 4 copies / ml, 10 3 (copies / ml), each gradient sample was repeated at least 5 times, and a standard curve experiment was performed. The slope, R value, and amplification efficiency of the standard curve were statistically analyzed. The amplification curves for monkeypox virus and monkey B virus are shown below. Figure 1 and Figure 3 The standard curves for monkeypox and monkey B virus are as follows: Figure 2 and Figure 4 The slope of the monkeypox virus correlation coefficient was -3.492, and the correlation coefficient (R²) was [missing value]. 2 The amplification efficiency (E = 10) was 0.999. ∧ The (-1 / K)-1) is 93.345%. The slope of simian B virus is -3.383, and the correlation coefficient (R) is... 2 The amplification efficiency (E = 10) was 0.996. ∧ (-1 / K)-1) is 97.475%.

[0049] V. Sensitivity Test

[0050] The lowest concentration standard (10) in the monkeypox and monkey B virus standard curve was used. 3The samples (copies / ml) were diluted to 500 copies / ml using a 2-fold serial dilution method. These two concentration gradients were tested 20 times each, and the positive detection rate for each gradient was calculated. The lowest gradient sample achieving a 95% positive detection rate was taken as the limit of detection (LOD) of the kit. The results are shown in Tables 3 and 4.

[0051] Table 3 Monkeypox virus sensitivity test

[0052] Template concentration Number of tests CT mean Detection rate <![CDATA[10 3 copies / ml]]> 20 37.1 100％ 500 copies / ml 20 38.3 100％

[0053] Table 4. Monkey B Virus Sensitivity Test

[0054] Template concentration Number of tests CT mean Detection rate <![CDATA[10 3 copies / ml]]> 20 36.6 100％ 500 copies / ml 20 37.9 100％

[0055] VI. Melting Curve Analysis

[0056] The principle of the melting curve method in this invention's kit is to perform specific PCR amplification of the genes of monkeypox virus and monkey B virus, and then generate melting curves. Because the amplicon size and GC content of monkeypox virus and monkey B virus differ significantly, the Tm values ​​of the melting curves for different viruses will shift. The type of virus infection can be determined by the Tm value of the melting curve. The Tm values ​​for monkeypox virus and monkey B virus are 73.92℃ and 79.71℃, respectively. The melting curves for monkeypox virus and monkey B virus are shown below. Figure 5 .

[0057] <Example>

[0058] A method for simultaneously detecting monkeypox virus and monkey B virus includes the following steps:

[0059] 1) Specific PCR amplification of monkeypox virus and monkeypox B virus; the amplification system for monkeypox virus and monkeypox B virus was as follows: PCR buffer 10 μL, primers for monkeypox virus and monkeypox B virus 5 μL each, primers SEQ ID No. 01 (MPVF) and SEQ ID No. 02 (MPVR) at 0.4 μM, primers SEQ ID No. 03 (MBVF) and SEQ ID No. 04 (MBVR) at 0.35 μM, fluorescent dye SYBR Green 5 μL; positive standard or negative control or sample format extraction solution 5 μL, deionized water 5 μL; the amplification program for monkeypox virus and monkeypox B virus was as follows: 95℃ pre-denaturation for 20 seconds; 95℃ denaturation for 5 seconds; 60℃ annealing for 20 seconds, for a total of 40 cycles;

[0060] 2) After amplification, a melting curve is generated to distinguish between monkeypox virus and monkey B virus by using the difference in Tm value of the melting curve. That is, the Tm value of 73.92℃ is monkeypox virus, and the Tm value of 79.71℃ is monkey B virus.

[0061] The fluorescent dye can be any one of SYBR Green, Eva Green, Helix Green, and LC Green.

[0062] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A fluorescent PCR kit for simultaneous detection of monkeypox and monkey B virus, characterized in that, The kit contains a mixture of primers for the simultaneous detection of monkeypox virus and monkey B virus. The primer sequences for detecting monkeypox virus are shown in SEQ ID No. 01 and SEQ ID No. 02; and the primer sequences for detecting monkeypox virus are shown in SEQ ID No. 03 and SEQ ID No.

04.

2. The fluorescent PCR kit for simultaneous detection of monkeypox and monkey B virus according to claim 1, characterized in that, The kit also contains a nonspecific fluorescent dye, which is one of SYBR Green, Eva Green and LCGreen.

3. The fluorescent PCR kit for simultaneous detection of monkeypox and monkey B virus according to claim 1, characterized in that, The kit also includes a positive control and a negative control, wherein the positive control is prepared from a plasmid mixture of monkeypox virus and monkey B virus, and the negative control is prepared from TE buffer.

4. The method for simultaneously detecting monkeypox and monkey B virus using the fluorescent PCR kit according to any one of claims 1-3 for non-disease diagnosis, characterized in that, Includes the following steps: 1) Specific PCR amplification of monkeypox virus and monkeypox B virus; 2) After amplification, a melting curve is generated to distinguish between monkeypox virus and monkey B virus by using the difference in Tm values ​​of the melting curve.

5. The method for simultaneously detecting monkeypox and monkey B virus using the fluorescent PCR kit as described in claim 4 for non-disease diagnosis, characterized in that... The amplification system for monkeypox virus and monkeypox B virus is as follows: PCR buffer 10-20uL, primers for monkeypox virus and monkeypox B virus 3-5uL each, positive standard or negative control or sample nucleic acid extract 3-5uL, and deionized water 5-10uL.

6. The method for detecting monkeypox and monkey B virus simultaneously using the fluorescent PCR kit as described in claim 5 for non-disease diagnosis, characterized in that... The primer concentration used for amplifying monkeypox virus was 0.4 μM, and the primer concentration used for amplifying monkey B virus was 0.35 μM.

7. The method for simultaneously detecting monkeypox and monkey B virus using the fluorescent PCR kit as described in claim 5 for non-disease diagnosis, characterized in that... The amplification program for monkeypox virus and monkeypox B virus is as follows: pre-denaturation at 95℃ for 20 seconds; denaturation at 95℃ for 5 seconds; annealing at 56-62℃ for 20 seconds, for a total of 40-45 cycles.

8. The method for detecting monkeypox and monkey B virus simultaneously using the fluorescent PCR kit as described in claim 7 for non-disease diagnosis, characterized in that, The amplification program for monkeypox virus and monkeypox B virus was as follows: 95℃ pre-denaturation for 20 seconds; 95℃ denaturation for 5 seconds; 60℃ annealing for 20 seconds, for a total of 40 cycles.

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