A lAMP detection kit for detecting monkey b virus

Abstract

The present application relates to a kind of LAMP detection kit for detecting monkey B virus, belong to biological medicine technical field.The present application provides a kind of diagnostic kit for detecting monkey B virus, kit is used to detect the UL30 gene sequence of monkey B virus;Kit includes nucleotide sequence such as SEQ ID No.1 to SEQ ID No.6, SEQ ID No.7 to SEQ ID No.12, SEQ ID No.13 to SEQ ID No.18, SEQ ID No.19 to SEQ ID No.24 or SEQ ID No.25 to SEQ ID No.30 as shown in the primer and probe combination.Probe end base is labeled with fluorescent group and quencher group respectively.By the present application, a kind of kit can be quickly and accurately diagnosed monkey B virus;It is simple, fast and convenient to use, suitable for grass-roots rapid diagnosis.

Description

Technical Field

[0001] This invention relates to a LAMP detection kit for detecting monkey B virus, belonging to the field of biomedical technology. Background Technology

[0002] Herpesvirus B, also known as simian herpesvirus, is referred to as cercopithecine herpesvirus 1 by the International Committee on Taxonomy of Viruses (ICVs) due to its numerous names. It belongs to the genus *Herpessimus simplex* within the subfamily Alphaherpesvirinae of the family Herpesviridae. Herpesvirus B is a double-stranded DNA virus with an envelope, a diameter of 160–180 mm, and multiple open reading frames. Some of its amino acid sequences share approximately 79% homology with HSV-1 and HSV-2. Herpesvirus B exhibits a typical herpesvirus structure, with an icosahedral capsid enclosing a viral DNA core.

[0003] Monkey B virus infection primarily occurs in rhesus monkeys and cynomolgus monkeys, commonly used in biomedical experiments. Monkeys are typically infected between 2 and 4 years of age, reaching sexual maturity. The seropositivity rate gradually increases with age, reaching 80%–100% in adult monkeys. Infected monkeys generally carry the virus for life, but show little or no clinical symptoms. Sometimes, they may exhibit herpes-like lesions in the oral cavity, such as oral ulcers or conjunctivitis. Genital tract lesions are absent in monkeys. Monkeys shed the virus through saliva and the genital tract. Transmission between monkeys typically occurs through mating, bites, or scratches. The pathogenesis of monkey B virus infection is similar to that of HSV (herpes simplex virus). The virus first multiplies in epithelial tissue and then invades nerve cells. Neutralizing antibodies against B virus generally appear in monkey serum about 3 weeks after infection. Pathological changes include the appearance of blisters on the dorsum of the tongue, and the gradual rupture of blisters on the lips or other mucous membranes, resulting in vesicle scars, which usually heal within 2 weeks. Severe keratitis may also occur in monkeys. Most cases of simian B virus infection are related to direct contact with monkeys, such as scratches, bites, or contact with mucous membranes through the monkey's bodily fluids or secretions. Indirect contact mainly involves scratches from monkey cages or pricks from contaminated needles. Once infected, the mortality rate in humans is as high as 70%. Early and rapid diagnosis of simian B virus infection is crucial for the prevention and control of this disease. Currently used detection methods mainly include the following: 1) Isolation and culture of simian B virus is the standard diagnostic method for infection and the only method for quantitative detection of simian B virus in clinical samples. However, this virus belongs to Biosafety (BSL) Level 4 pathogens, and its culture requires a minimum of BSL-3 facilities; 2) Perelygina et al. determined the complete gene sequence of simian B virus strain E2490 and studied its gene structure and function. They designed specific primers and amplified conserved target genes that do not cross-link with viruses such as HSV1 by PCR to detect B virus DNA; 3) In serological detection of simian B virus, using alternative antigens, such as simian factor 8 (SA8) and baboon herpesvirus, can reduce the risks of handling simian B virus. However, the detection sensitivity is significantly lower than that of detection methods established using the original virus as the antigen. In summary, most existing methods for detecting simian B virus have long detection cycles, are slow, and have low accuracy. Therefore, there is an urgent need in this technical field for a product that can achieve rapid, effective, and accurate detection of simian B virus.

[0004] Loop-mediated isothermal amplification (LAMP) is characterized by the design of four specific primers targeting six regions of the target gene. Under the action of strand displacement DNA polymerase (Bst DNA polymerase), amplification is carried out at an isothermal temperature of 60–65°C, achieving 10-1 amplifications in approximately 15–60 minutes. 9 ~1010 This LAMP reaction, characterized by its high specificity, rapid nucleic acid amplification, and ease of product detection, offers several advantages. To further accelerate the reaction, two circular primers can be introduced. These primers complement the circular regions formed during synthesis, increasing the number of DNA synthesis origins in the LAMP reaction and significantly shortening detection time and improving efficiency. Besides its high specificity and sensitivity, this technique is remarkably simple to operate, requiring minimal equipment—a water bath or incubator is sufficient. Result identification is also straightforward; unlike PCR, gel electrophoresis is not necessary. The results of the loop-mediated isothermal amplification reaction are visually observed for the formation of white turbidity or green fluorescence, making it convenient, fast, and suitable for rapid diagnosis at the grassroots level. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problem of how to obtain a detection system that can quickly and accurately detect monkey B virus.

[0006] To address the aforementioned problems, the present invention provides a diagnostic reagent for detecting monkey B virus, wherein the reagent is used to detect the UL30 gene sequence of monkey B virus.

[0007] Preferably, the reagent comprises primer and probe combinations with nucleotide sequences such as those shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24, or SEQ ID No. 25 to SEQ ID No. 30.

[0008] Preferably, the bases at both ends of the probe are labeled with a fluorescent group and a quenching group, respectively.

[0009] The present invention provides a LAMP kit for detecting simian B virus, the kit comprising primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24, or SEQ ID No. 25 to SEQ ID No. 30.

[0010] Preferably, the LAMP kit is based on the LAMP method for detecting simian B virus.

[0011] This invention provides the application of a primer combination in the preparation of a detection kit for monkey B virus, wherein the primer combination comprises a primer and probe combination with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24 or SEQ ID No. 25 to SEQ ID No. 30.

[0012] This invention provides a method for detecting simian B virus, comprising detecting simian B virus based on LAMP method; the target gene to be detected is the UL30 gene sequence of simian B virus; the detection reagent includes primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24 or SEQ ID No. 25 to SEQ ID No. 30.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention provides a reagent kit and diagnostic method for rapid and accurate diagnosis of monkey B virus; the provided reagent kit is simple and quick to use, and is suitable for rapid diagnosis at the grassroots level.

[0015] The operation of this invention is simple and fast; the LAMP method established by this invention is faster and more efficient than conventional quantitative PCR and other monkey B virus detection methods. It can be completed within 40 minutes from sample extraction to result determination, and the reaction result determination method is simple.

[0016] The diagnostic kit provided by this invention has high sensitivity; the primers of this invention can achieve a sensitivity of 1 copy per microliter, and can be stably detected at concentrations of 10 copies / reaction or higher, which is significantly higher than the level of existing technology. Attached Figure Description

[0017] Figure 1 The results show the comparison of detection sensitivity of five primer sets (UL1, UL2, UL3, UL4, and UL5) for the UL30 gene at the same template concentration.

[0018] Figure 2 The results show the sensitivity detection of a single target in a single tube using the UL1 primer set isothermal amplification method. Detailed Implementation

[0019] To make the present invention more apparent and understandable, preferred embodiments are described in detail below:

[0020] The technical solution adopted by this invention is to provide a diagnostic reagent for detecting simian B virus, the reagent being used to detect the UL30 gene sequence of simian B virus; the reagent includes primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24, or SEQ ID No. 25 to SEQ ID No. 30; the bases at both ends of the probe are respectively labeled with a fluorescent group and a quencher group.

[0021] This invention provides a LAMP kit for detecting simian B virus, comprising primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24, or SEQ ID No. 25 to SEQ ID No. 30; the LAMP kit detects simian B virus based on the LAMP method.

[0022] This invention provides the application of a primer combination in the preparation of a detection kit for monkey B virus, wherein the primer combination comprises a primer and probe combination with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24 or SEQ ID No. 25 to SEQ ID No. 30.

[0023] This invention provides a method for detecting simian B virus, comprising detecting simian B virus based on LAMP method; the target gene to be detected is the UL30 gene sequence of simian B virus; the detection reagent includes primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6, SEQ ID No. 7 to SEQ ID No. 12, SEQ ID No. 13 to SEQ ID No. 18, SEQ ID No. 19 to SEQ ID No. 24 or SEQ ID No. 25 to SEQ ID No. 30.

[0024] Example 1

[0025] 1.1 LAMP primer set design:

[0026] Based on the conserved UL30 gene sequence of simian B virus reported in NCBI (accession number GenBank: AF533768.1), five specific primer sets (UL1, UL2, UL3, UL4, and UL5) were designed using the LAMP primer design software Primer Explorer V5 (http: / / primerexplorer.jp / lampv5e / index.html). The primer and fluorescently labeled probe sequences (5'-3') are shown in Table 1 below:

[0027] Table 1. Primer set for monkey B virus detection

[0028]

[0029]

[0030] 1.2 Screening of LAMP primers:

[0031] The five primer sets designed for the dual targets in Example 1.1 were optimized through sensitivity comparison. The specific method is as follows: Based on the five primer sets designed in Example 1.1, isothermal amplification systems were prepared using each of the five primer sets. The plasmid pcDNA3.1(+) (synthesized by Sangon Biotech) containing the target UL30 gene (GenBank: AF533768.1) was diluted using a NanoDrop One / OneC micro-nucleic acid protein concentration analyzer to determine its concentration and then serially diluted to 10⁻⁶. 3 3 μL of the template (copy / μL) was added to the isothermal amplification system of five primer sets for reaction. The reaction conditions were 63℃ for 1 min / 40 cycles. The amplification efficiency of each primer set was compared under the same conditions.

[0032] The LAMP isothermal amplification reaction system is shown in Table 2 below.

[0033] Table 2. LAMP isothermal amplification reaction system

[0034]

[0035]

[0036] LAMP isothermal amplification reaction conditions:

[0037] React at 50-65℃ for 10-60 minutes, with the optimal reaction conditions being 64℃ for 40 minutes.

[0038] The sensitivity verification results of the 5 primer sets are as follows: Figure 1 ,Depend on Figure 1It can be seen that, under the same template concentration, the UL1 primer set has the shortest time to produce an amplification curve compared to other primer sets. Therefore, UL1 is the preferred primer set, and this primer set will be used in the following experiments.

[0039] Example 2

[0040] Results of sensitivity testing for the UL1 primer set isothermal amplification method:

[0041] After primer screening, the concentration of the simian B virus genome standard material (plasmid pcDNA3.1(+) containing the target UL30 gene (GenBank: AF533768.1), synthesized by Sangon Biotech) was determined using a NanoDrop One / OneC micro-nucleic acid protein concentration analyzer. The sample was then serially diluted 10-fold to 1000 copies / μL, 100 copies / μL, 10 copies / μL, and 1 copy / μL. Using samples from all dilutions as templates, amplification was performed using the LAMP detection kit of this invention.

[0042] The specific operation of the isothermal amplification detection method is as follows: Taking a 25 μL isothermal amplification system as an example, the following are the components: 2.5 μL of 10X isothermal amplification system buffer (New England Biolabs, MA), 1 μL of 8 U / μL Bst DNA polymerase (New England Biolabs Inc., MA), 1 μL of 100 mM MgSO4, 3.5 μL of 10 mM dNTP, 0.5 μL of 10 μM primer F3, 0.5 μL of 10 μM primer B3, 0.5 μL of 80 μM primer FIP, 0.5 μL of 80 μM primer BIP, 0.5 μL of 40 μM primer LF, and 40 μM... 0.5 μL of LB primer (OPG002 or ATI primer set), 5 μL of betaine (Sigma-Aldrich, MO), 3 μL of template, and nuclease-free water to a final volume of 25 μL. Reaction program: 64 °C, 40 min, single-tube single-target reaction.

[0043] Test results as follows Figure 2 As shown. The positive and negative control test results were normal. All other concentrations in this application were detected, with a detection probability of 1 copy / μL. Based on a sample volume of 3μL, concentrations of 10 copies / reaction or higher can be stably detected.

[0044] Therefore, the LAMP detection kit and detection method for simian B virus provided by this invention can complete the detection of simian B virus strains within 40 minutes. The detection speed is fast, the operation is simple, and it can be widely used for rapid identification and diagnosis of simian B virus strains.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A diagnostic reagent for detecting simian B virus, characterized in that, The reagent is used to detect the UL30 gene sequence of simian B virus; The reagents include primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No.

6.

2. The diagnostic reagent for detecting simian B virus according to claim 1, characterized in that, The probe has a fluorescent group and a quenching group labeled at both ends.

3. A LAMP kit for detecting simian B virus, characterized in that, The kit includes primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No.

6.

4. The LAMP kit for detecting simian B virus according to claim 3, characterized in that, The LAMP kit is based on the LAMP method for detecting monkey B virus.

5. The application of a primer combination in the preparation of a detection kit for monkey B virus, characterized in that, The primer combination includes primer and probe combinations with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 6.

Images