Compositions, kits, methods of detecting monkeypox virus and uses thereof

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to a composition, a kit, a method for detecting monkeypox virus and typing and use thereof. The present application provides a composition for detecting monkeypox virus and typing, and simultaneously provides a kit comprising the composition, use of the composition, and a method for detecting monkeypox virus and typing. Using the composition of the present application, monkeypox virus can be rapidly detected with a sensitivity of 200 copies / mL. The detection and typing of monkeypox virus are simultaneously achieved in a reaction system. Different monkeypox branches can be distinguished, and treatment and prevention can be more targeted. The composition of the present application can further increase the target, detect more targets in a single tube, more greatly avoid the possibility of false negatives, and improve the accuracy of detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically relating to compositions, kits, methods, and uses for detecting and typing monkeypox virus. Background Technology

[0002] Monkeypox is a zoonotic viral disease caused by infection with the monkeypox virus (MPXV).

[0003] The incubation period for monkeypox is 5-21 days, mostly 6-13 days. Early symptoms include chills and fever, with temperatures often above 38.5℃, accompanied by headache, drowsiness, fatigue, back pain, and muscle pain. Most patients experience swollen lymph nodes in the neck, armpits, and groin. A rash appears 1-3 days after onset. The rash first appears on the face and gradually spreads to the limbs and other areas, often exhibiting a centrifugal distribution. The rash is more common on the face and limbs than on the trunk, and can appear on the palms and soles. The number of rashes can range from a few to thousands; it can also affect the oral mucosa, digestive tract, genitals, conjunctiva, and cornea. The rash progresses through several stages: macules, papules, vesicles, pustules, and finally crusting. Vesicles and pustules are usually spherical, about 0.5-1 cm in diameter, firm in texture, and may be accompanied by significant itching and pain. The time from onset to crusting and shedding is approximately 2-4 weeks. After the scab falls off, erythema, pigmentation, or even scarring may remain, and the scars can last for years. Some patients may experience complications, including secondary bacterial infections at the site of the lesion, bronchopneumonia, encephalitis, corneal infection, sepsis, etc.

[0004] Monkeypox is a self-limiting disease with a generally good prognosis. Severe cases are common in young children and immunocompromised individuals, and prognosis is related to the viral lineage, the degree of viral exposure, pre-existing health conditions, and the severity of complications. Monkeypox virus has two distinct genetic clades: clade I (proto-Congo Basin (Central African) clade) and clade II (proto-West African clade), with clade II further divided into clades IIa and IIb. The mortality rate of clade II (proto-West African clade) is approximately 3%, while that of clade I (proto-Congo Basin (Central African) clade) is approximately 10%. This demonstrates a significant difference in lethality between the two lineages. Since the epidemiological and clinical characteristics of the disease caused by clade I (proto-Congo Basin (Central African) clade) and clade II (proto-West African clade) differ, strengthening the characterization of genetic differences between these two lineages and accurately typing clade I (proto-Congo Basin (Central African) clade) and clade II (proto-West African clade) is essential for disease control.

[0005] Currently, the main diagnostic methods for viral infections, both domestically and internationally, are serological tests, viral culture, and PCR. Among these, serological tests and tissue culture have drawbacks such as low sensitivity, cross-reactivity with immune systems, and long processing times.

[0006] Therefore, there is a need in the art for a highly sensitive and rapid diagnostic product for monkeypox virus that can genotype the West African and Democratic Republic of Congo lineages in order to respond to sudden monkeypox outbreaks. Summary of the Invention

[0007] In view of this, in a first aspect, the present invention provides a composition for detecting monkeypox virus, the composition comprising:

[0008] First nucleic acid composition:

[0009] As shown in SEQ ID NO:1~3, universal upstream primers, universal downstream primers, and universal probes for detecting monkeypox virus; or

[0010] As shown in SEQ ID NO:4~6, universal upstream primer, universal downstream primer, and universal probe for detecting monkeypox virus;

[0011] Second nucleic acid composition:

[0012] For example, the upstream primers, downstream primers, and probes for detecting the West African branch of monkeypox virus shown in SEQ ID NO:7~9; or

[0013] As shown in SEQ ID NO:10~12, the upstream primer, downstream primer, and probe for detecting the West African branch of monkeypox virus; and

[0014] Third nucleic acid composition:

[0015] As shown in SEQ ID NO:13~15, the upstream primer, downstream primer, and probe for detecting the DRC branch of monkeypox virus; or

[0016] The upstream primer, downstream primer, and probe for detecting the Congo branch of monkeypox virus are shown in SEQ ID NO:16~18.

[0017] Using the composition of this invention, monkeypox virus can be rapidly diagnosed and genotyped in a single tube with a sensitivity of 200 copies / mL, simultaneously detecting and genotyping the West African and Democratic Republic of Congo lineages. This allows for differentiated treatment of different strains, leading to more efficient treatment and prevention. The composition of this invention is low-cost, high-throughput, and easy to operate; results can be read through different channels. The entire detection process is conducted under closed conditions, avoiding false positives and environmental contamination caused by cross-contamination between samples.

[0018] In some specific embodiments, at least one of the first, second, and third nucleic acid compositions of the present invention includes two pairs of primers and probes.

[0019] In some specific embodiments, at least two of the first, second, and third nucleic acid compositions of the present invention include two pairs of primers and probes.

[0020] In some specific implementations, the first, second, and third nucleic acid compositions of the present invention each include two pairs of primers and probes.

[0021] For example, in one specific embodiment, the present invention provides a composition for detecting monkeypox virus, the composition comprising:

[0022] First nucleic acid composition:

[0023] As shown in SEQ ID NO:1~3, universal upstream primer, universal downstream primer, and universal probe for detecting monkeypox virus;

[0024] As shown in SEQ ID NO:4~6, universal upstream primer, universal downstream primer, and universal probe for detecting monkeypox virus;

[0025] Second nucleic acid composition:

[0026] As shown in SEQ ID NO:7~9, upstream primers, downstream primers, and probes for detecting the West African branch of monkeypox virus.

[0027] As shown in SEQ ID NO:10~12, the upstream primer, downstream primer, and probe for detecting the West African branch of monkeypox virus; and

[0028] Third nucleic acid composition:

[0029] As shown in SEQ ID NO:13~15, upstream primers, downstream primers, and probes for detecting the DRC branch of monkeypox virus.

[0030] The upstream primer, downstream primer, and probe for detecting the Congo branch of monkeypox virus are shown in SEQ ID NO:16~18.

[0031] Using the composition described above, the number of targets can be further increased, more targets can be detected in a single tube, the possibility of false negatives can be avoided to a greater extent, and the accuracy of detection can be improved.

[0032] Furthermore, in some embodiments, the composition of the present invention may simultaneously include one or more pairs of the primer and probe pairs described above. In the present invention, a "pair" refers to a mutually matched upstream and downstream primer and probe for detecting a target.

[0033] The compositions of this invention can be arbitrarily combined to detect any combination of the nine target sites. Those skilled in the art can combine them as needed, determining which target sites to detect by combining the primer and probe pairs corresponding to those target sites. All such combinations are included in this invention.

[0034] For example, it may include any 8 pairs of the above 9 pairs of primers and probes, any 7 pairs of the above 9 pairs of primers and probes, any 6 pairs of the above 9 pairs of primers and probes, any 5 pairs of the above 9 pairs of primers and probes, any 4 pairs of the above 9 pairs of primers and probes, any 3 pairs of the above 9 pairs of primers and probes, any 2 pairs of the above 9 pairs of primers and probes, or any 1 pair of the above 9 pairs of primers and probes.

[0035] In some specific embodiments, the composition of the present invention further includes primers and probes for detecting internal standards.

[0036] Furthermore, the fluorescent groups of different nucleic acid compositions are different from each other and do not interfere with each other.

[0037] In this article, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorescent group, and these groups do not affect each other's detection; that is, different channels can be used for detection. For example, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values, and different channels can be selected, so they will not interfere with each other.

[0038] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0039] In this invention, the fluorescent reporter group may be selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 and JOE, but is not limited thereto.

[0040] In some specific embodiments, the fluorescent group of the probe shown in SEQ ID NO:3 is FAM; the fluorescent group of the probe shown in SEQ ID NO:6 is VIC; the fluorescent group of the probes shown in SEQ ID NO:9 and 12 is CY5; and the fluorescent group of the probes shown in SEQ ID NO:15 and 18 is ROX.

[0041] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1, BHQ2, or MGB.

[0042] In one specific implementation, the 3' end of the probe is BHQ1.

[0043] In one specific implementation, the 3' end of the probe is an MGB.

[0044] Further, the amount of primers used in the composition is 0.05 μM to 0.5 μM; the amount of probes used in the composition is 0.05 μM to 0.5 μM.

[0045] In one specific embodiment, each nucleic acid composition of the present invention is contained in a separate package.

[0046] In one specific embodiment, the nucleic acid compositions of the present invention exist in a mixed form.

[0047] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for detecting and typing monkeypox virus.

[0048] Thirdly, the present invention provides a kit for detecting and typing monkeypox virus, the kit comprising the composition of the present invention as described above.

[0049] Furthermore, the kit also includes negative and positive controls.

[0050] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0051] Furthermore, the kit also includes at least one of the following: a nucleic acid release agent, a nucleic acid extraction reagent, a uracil glycosylation enzyme, and a DNA polymerase.

[0052] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, uracil glycosylase, DNA polymerase, PCR buffer, and Mg... 2+ At least one of them.

[0053] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase can be Taq polymerase.

[0054] In one specific embodiment, the kit of the present invention comprises: Taq enzyme, uracil glycosylation enzyme, and Mg... 2+ Mn 2 + dNTPs, primers, probes, and PCR buffer.

[0055] Common PCR buffers consist of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. The total volume in a single PCR reaction tube is typically 20µL to 100µL.

[0056] In one specific implementation, the kit of the present invention is compatible with digital PCR amplification systems, that is, it can be directly used for amplification on a digital PCR instrument.

[0057] Fourthly, a method for detecting and typing monkeypox virus is provided, the method comprising the following steps:

[0058] 1) Extract or release nucleic acid from the sample to be tested;

[0059] 2) Perform quantitative real-time PCR analysis on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0060] 3) Obtain and analyze the results.

[0061] In some specific implementations, the sample is whole blood, vesicles, or pustules.

[0062] In yet another specific embodiment, the present invention provides a method for detecting and typing monkeypox virus for non-diagnostic purposes, the method comprising the following steps:

[0063] 1) Extract or release nucleic acid from the sample to be tested;

[0064] 2) Perform quantitative real-time PCR analysis on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0065] 3) Obtain and analyze the results.

[0066] In some specific implementations, the sample is whole blood, vesicles, or pustules.

[0067] In this article, the term "non-diagnostic purpose" refers to a method not intended to obtain information about whether an individual is infected with monkeypox virus. For example, this method can be used to detect the presence and typing of monkeypox virus in test cultures used in experiments for research purposes. Attached Figure Description

[0068] Figure 1 The detection results of universal monkeypox virus (DRC branch) for the composition of the present invention.

[0069] Figure 2 The detection results (West African branch) of the universal monkeypox virus for the composition of the present invention.

[0070] Figures 3-4 The results of sensitivity testing of the composition of the present invention;

[0071] Figure 5 This is the result of specific detection of the composition of the present invention;

[0072] Figures 6-8 The results of detecting various viral branches using the comparative composition of this invention are shown.

[0073] Figures 9-12 The results of single-target and dual-target detection of various viral branches using the composition of the present invention are shown (dual-target detection of the Democratic Republic of Congo, single-target detection of the Democratic Republic of Congo, dual-target detection of West Africa, and single-target detection of West West Africa, respectively). Detailed Implementation

[0074] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0075] Example 1: Primers and probes used in this invention

[0076] Table 1

[0077]

[0078] Among them, the fluorescent group of the probe shown in SEQ ID NO:3 is FAM; the fluorescent group of the probe shown in SEQ ID NO:6 is VIC; the fluorescent group of the probes shown in SEQ ID NO:9 and 12 is CY5; and the fluorescent group of the probes shown in SEQ ID NO:15 and 18 is ROX.

[0079] Example 2: Method for detecting and typing monkeypox virus

[0080] Reagent test kit:

[0081] (1) qPCR reaction solution: MgCl2 (3-8mM), dNTPs (0.5-1.0mM), and upstream / downstream primer concentrations of 0.1-0.3μM, probe concentration of 0.1-0.3μM, universal upstream / downstream primer 2-6μM, hot-start Taq enzyme, uracil glycosylation enzyme (UNG);

[0082] (3) Negative control: DEPC H2O.

[0083] (4) Positive control: a mixture of fake viruses.

[0084] The detection method is as follows:

[0085] 1. Specimen types: Skin lesion specimens (including swabs of lesions, vesicle surface and / or exudate, vesicle fluid, vesicle epidermis or scabs, etc.), throat swabs, whole blood or serum samples.

[0086] 2. Nucleic acid extraction:

[0087] Commercial DNA extraction kits, such as nucleic acid extraction reagents based on silica membrane centrifugation column method or nucleic acid extraction reagents based on magnetic bead method, were used and operated according to the kit instructions. Finally, 70 μL of DNA solution was collected and directly detected.

[0088] Alternatively, store at -80℃. Both negative and positive quality controls must be extracted.

[0089] 3. System Configuration:

[0090] Add 15 μl of qPCR reaction solution to each PCR tube according to the total number of reactions N required for the test. Calculate the required total volume, mix well, and then aliquot into specially designed PCR reaction tubes.

[0091] 4. Sample addition:

[0092] Add 10 μL of negative control, sample DNA solution, and positive control to the PCR reaction tubes that have been aliquoted with reagents. Tighten the caps, mix well, centrifuge, and collect the solution at the bottom of the tube.

[0093] 5. Amplification and detection on the instrument:

[0094] The settings for the qPCR amplification program and melting curve analysis program are as follows:

[0095]

[0096] 6. Results Analysis:

[0097] Assuming effective amplification, the criteria for determining positive Ct values ​​for each target gene are as follows:

[0098]

[0099] Example 3: Detection results of test samples of the composition of the present invention

[0100] The primers and probes shown in Example 1 were validated against monkeypox virus gene plasmid samples according to the method in Example 2. The results showed accurate detection, indicating that the composition of the present invention can accurately identify and diagnose monkeypox virus. Figure 1 and 2 As shown, the composition of the present invention can also accurately type West African and Central African branch monkeypox viruses.

[0101] Example 4: Sensitivity of the composition of the present invention

[0102] The following pseudovirus standards were purchased from the National Institute of Metrology of China: monkeypox virus mutant F3L gene pseudovirus standard (NIM-RM4060), monkeypox virus wild-type B6R gene pseudovirus standard (NIM-RM4059), pseudovirus containing the target gene sequence from branch I (former Congo Basin (Central Africa) branch), and monkeypox virus branch II (former West Africa branch). These were mixed with negative samples such as throat swabs, whole blood, serum, and vesicular fluid, diluted to a concentration of 200 copies / mL. These were used to simulate positive samples from branch I (former Congo Basin (Central Africa) branch) and monkeypox virus branch II (former West Africa branch), respectively. The lowest detection limit reference was extracted and detected according to the kit instructions. The detection was repeated three times to verify the sensitivity of the reagent and detection method. The test results are shown in the figure below. Monkeypox virus, diluted in negative matrices such as throat swabs, whole blood, serum, and vesicular fluid at a concentration of 200 copies / mL, accurately detected at 200 copies / mL in simulated branch I (proto-Congo Basin (Central African) branch) and branch II (proto-West African branch). This indicates that the detection method has high sensitivity. Some test results are shown below. Figures 3-4 As shown.

[0103] Example 5: Specificity of the composition of the present invention

[0104] The human genomic DNA of 25 pathogens, including smallpox virus (pseudovirus), vaccinia virus, cowpox virus, mousepox virus, molluscum contagiosum virus, Turner pox virus (pseudovirus), maculopapular virus (pseudovirus), varicella-zoster virus, rubella virus, herpes simplex virus-1 / -2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, measles virus, enterovirus, Treponema pallidum, dengue virus, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Candida albicans, Propionibacterium acnes, and Corynebacterium diphtheriae, was used as a specific detection sample for testing. The test results are shown in the figure. The test results showed that 25 pathogens, including smallpox virus (pseudovirus), vaccinia virus, cowpox virus, mousepox virus, molluscum contagiosum virus, Turner pox virus (pseudovirus), maculopapular virus (pseudovirus), varicella-zoster virus, rubella virus, herpes simplex virus-1 / -2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, measles virus, enterovirus, Treponema pallidum, dengue virus, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Candida albicans, Propionibacterium acnes, and Corynebacterium diphtheriae, as well as human genomic DNA, all tested negative, indicating that this kit has good specificity. Figure 5 As shown.

[0105] Comparative Example 1: Other primers and probes designed in this invention that do not perform well.

[0106] Due to the complementary base pairing principle, primers and / or probes can form dimers, but this probability is low and can be eliminated at the initial design stage. However, when detecting multiple pathogens together, there are numerous primers and probes, and dimers can easily form between primers, probes, or between different primers and probes. To ensure the conservation of the design (conservatism is crucial for detection accuracy) while also considering the mutual interference between different primers and probes, careful primer and probe design is required.

[0107] Therefore, the inventors also designed other primers and probes (sequences not shown) to form different detection systems 1, 2, and 3, which were also used to detect and genotype monkeypox virus. Specific detection results are as follows: Figures 6-8 As can be seen from the figure, only some amplification curves were detected, and the amplification curves showed low amplification and poor repeatability. Other targets did not even have amplification curves. Therefore, the overall detection effect was poor.

[0108] Comparative Example 2: Comparison of detection effects between single-target and dual-target targets

[0109] The inventors also designed a comparative test between single-target and dual-target genes, which was also used to detect and genotype monkeypox virus. Specific test results are as follows: Figures 9-12 As can be seen from the figure, under the premise of single-target gene detection, when the sample concentration is low, it is impossible to accurately genotype monkeypox virus branches, which may lead to missed detection of branch results and low genotyping accuracy. However, dual-target detection can accurately genotype low-concentration samples, reducing the probability of missed detection.

Claims

1. A composition for detecting monkeypox virus, the composition comprising: a first nucleic acid composition: a monkeypox virus universal upper primer, a monkeypox virus universal lower primer, a monkeypox virus universal probe as shown in SEQ ID NO: 1~3; a monkeypox virus universal upper primer, a monkeypox virus universal lower primer, a monkeypox virus universal probe as shown in SEQ ID NO: 4~6; a second nucleic acid composition: a monkeypox virus West African clade upper primer, a monkeypox virus West African clade lower primer, a monkeypox virus West African clade probe as shown in SEQ ID NO: 7~9; a monkeypox virus West African clade upper primer, a monkeypox virus West African clade lower primer, a monkeypox virus West African clade probe as shown in SEQ ID NO: 10~12; and a third nucleic acid composition: a monkeypox virus Democratic Republic of the Congo clade upper primer, a monkeypox virus Democratic Republic of the Congo clade lower primer, a monkeypox virus Democratic Republic of the Congo clade probe as shown in SEQ ID NO: 13~15; a monkeypox virus Democratic Republic of the Congo clade upper primer, a monkeypox virus Democratic Republic of the Congo clade lower primer, a monkeypox virus Democratic Republic of the Congo clade probe as shown in SEQ ID NO: 16~18.

2. The composition of claim 1, wherein, Each component of the composition is present in a mixed form.

3. Use of the composition of claim 1 or 2 in the preparation of a kit for detecting monkeypox virus and typing West African clade and Democratic Republic of the Congo clade.

4. A kit for detecting monkeypox virus and typing West African clade and Democratic Republic of the Congo clade, the kit comprising the composition of claim 1 or 2.

5. The kit of claim 4, wherein, The kit also includes at least one of a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs, uracil glycosylase, a DNA polymerase, a PCR buffer, and Mg 2+ ​ 6. A method for detecting monkeypox virus and typing West African clade and Democratic Republic of the Congo clade for non-diagnostic purposes, the method comprising the following steps: 1) extracting or releasing nucleic acid from a sample to be tested; 2) performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition of claim 1 or 2 or the kit of claim 4 or 5; 3) obtaining and analyzing the results.

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