Nest MIRA primer combination for detecting hantavirus HTNV type and SEOV type

Abstract

The invention discloses a nested MIRA primer combination for detecting hantavirus HTNV type and hantavirus SEOV type. The nested MIRA primer combination comprises a peripheral primer, an inner primer and a probe for detecting the hantavirus HTNV type and the hantavirus SEOV type, the peripheral primers for the hantavirus HTNV type are as shown in SEQ ID NO: 1 and SEQ ID NO: 2, the inner primers are as shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the probe is as shown in SEQ ID NO: 5; the peripheral primers for the hantavirus SEOV type are as shown in SEQ ID NO: 6 and SEQ ID NO: 7, the inner primers are as shown in SEQ ID NO: 8 and SEQ ID NO: 9, and the probe is as shown in SEQ ID NO: 10; the primer disclosed by the invention can complete nested multienzyme constant-temperature nucleic acid rapid amplification within 30 minutes at the temperature of 39 DEG C, is simple and convenient to operate, has lower requirements on instruments, equipment and professional skills of operators, and can achieve the lower detection limit of 14.2 copies / mu L.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and in particular, relates to a nested MIRA primer combination and a detection method for rapid detection of Hantavirus HTNV type and SEOV type. Background Art

[0002] Hantavirus belongs to the Bunyaviridae family. It is an enveloped, segmented, negative-strand RNA virus whose genome contains three RNA segments (S, M, and L), which encode nucleocapsid protein (NP, about 50 kDa), glycoprotein (Gn and Gc), and RNA-dependent RNA polymerase (L protein), respectively. Hantavirus infection is mainly manifested by prodromal symptoms such as fever and headache, followed by the development of acute respiratory failure characterized by non-cardiogenic pulmonary edema and a high mortality rate (52.4% to 78.0%). Severe patients usually die within 3-7 days, while survivors recover rapidly without obvious sequelae. Its pathogenesis mainly involves the direct pathogenic effect of the virus, and the kidney is the main organ damaged by early viral infection.

[0003] Hantaviruses are mainly divided into two categories: one can cause hemorrhagic fever with renal syndrome (HFRS), including Hantaan virus (HTNV), Seoul virus (SEOV) and Puumala virus (PUUV); the other can cause Hantavirus pulmonary syndrome (HPS), including unnamed virus (SNV), New York virus (NYV), dirty black creek virus (BCCNV) and oxbow lake virus (BAYV). Its main transmission route is inhalation of aerosols formed by the excrement or secretions of infected rodents, or eating food contaminated by it. Therefore, rapid and accurate detection of Hantavirus, especially HTNV and SEOV, is crucial.

[0004] Hantavirus detection methods mainly include virus isolation, serological testing and genetic testing. Genetic detection methods, such as RT-PCR and RT-qPCR, have better sensitivity and specificity than serological testing. Clinically, the diagnosis of Hantavirus infection relies on exposure history, clinical symptoms and serological experiments (such as immunofluorescence and ELISA). The latter assesses viral replication by detecting HTNV IgM or IgG antibody levels, but there is cross-reactivity. In the early stages of hemorrhagic fever with renal syndrome (HFRS) (within 12 days of onset), HTNV RNA can be detected in plasma. Although RT-PCR and RT-qPCR are the main molecular diagnostic methods for detecting HTNV RNA, their reliance on expensive instruments and professionals and the complexity of operation limit their application in rapid on-site diagnosis.

[0005] In recent years, the new generation of point-of-care molecular diagnostic technology (POCT) has developed rapidly, among which the multiple-enzyme isothermal rapid amplification technology (MIRA) has attracted much attention. MIRA technology has been widely used in the rapid diagnosis of infectious diseases due to its simplicity, sensitivity and accuracy. This technology uses a special recombinase (similar in function to T4 UvsX, but derived from Streptomyces coelicolor) to improve the amplification efficiency. Compared with other isothermal amplification technologies, it has higher stability and anti-interference ability. MIRA technology relies on the synergistic effect of multiple functional proteins, and can complete the rapid amplification of nucleic acids in only 5-20 minutes under the constant temperature of 37-42°C. Its detection has high sensitivity, strong specificity, low cost, fast speed and convenient operation, providing an efficient and reliable solution for the rapid typing detection of Hantavirus HTNV and SEOV types, making it an ideal choice for POCT in resource-limited areas. Summary of the invention

[0006] In order to solve the above problems existing in the detection of Hantavirus, the present invention provides a nested MIRA detection primer combination for Hantavirus HTNV type and SEOV type.

[0007] The nested MIRA primer combination for typing detection of Hantavirus HTNV type and Hantavirus SEOV type of the present invention comprises peripheral primers, inner primers and probes for detecting Hantavirus HTNV type and Hantavirus SEOV type;

[0008] The peripheral primers for Hantavirus HTNV type are shown in SEQ ID NO: 1 and SEQ ID NO: 2, the inner primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the probe is shown in SEQ ID NO: 5;

[0009] The peripheral primers for Hantavirus SEOV type are shown in SEQ ID NO:6 and SEQ ID NO:7, the internal primers are shown in SEQ ID NO:8 and SEQ ID NO:9, and the probe is shown in SEQ ID NO:10.

[0010] The probe is specially modified. The probe for the Hantavirus HTNV type is modified with a FAM fluorescent group at a position 30 bp away from the 5' end, replaced by THF at 33 bp, and modified with a BHQ1 group at 34 bp. The 3' end of the probe is modified with a C3Spacer group. The probe is:

[0011] CTGCTGTTTGACCACTTGGTTCYTCTATA / FAM-dT / CA / THF / / BHQ1-dT / RGCATTYACA T-C3Spacer;

[0012] The probe for Hantavirus SEOV type is modified with a FAM fluorescent group at 32 bp from the 5' end, replaced by THF at 34 bp, and modified with a BHQ1 group at 35 bp. The 3' end of the probe is modified with a C3 Spacer group. The probe is:

[0013] TGTATAATCACGGGTATACGGGTGGTTTTYA / FAM-dT / A / THF / / BHQ1-dT / GGGRAACA GGGT-C3 Spacer.

[0014] The nested MIRA assay using the above primer combination is as follows:

[0015] 1. Extract Hantavirus RNA using a viral RNA magnetic bead extraction kit;

[0016] 2. Prepare the nested MIRA reaction system, wherein the system comprises the above-mentioned primer combination for Hantavirus HTNV type and SEOV type;

[0017] The nested MIRA amplification reaction system is:

[0018]

[0019]

[0020] The reagents used in the above reaction system are all conventional commercially available reagents for nested MIRA amplification;

[0021] 3. The conditions for the nested MIRA amplification reaction were 39°C, and the peripheral MIRA reaction and the inner MIRA reaction time were 10 min and 20 min, respectively;

[0022] 4. Result judgment: The test result is determined based on the amplification curve. If an amplification curve appears, it is a positive test. If there is no amplification curve, it is a negative sample.

[0023] Advantages and technical effects of the present invention:

[0024] (1) Based on the nested MIRA technology, the present invention has developed a typing detection method for Hantavirus HTNV and SEOV. The method has the advantages of being convenient and rapid, and the result detection can be completed in 30 minutes, and can be applied to bedside detection;

[0025] (2) This method has the advantages of high sensitivity, strong specificity, and high accuracy, and the minimum detection limit for Hantavirus HTNV and SEOV types can reach 14.2 copies / μL;

[0026] (3) The detection method of the present invention has a short detection time, simple operation steps, no need for large-scale nucleic acid amplification equipment, low detection cost, and greatly saves manpower and material resources. It is of great significance for early diagnosis of diseases and observation of disease progression and guiding clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result of the internal primer screening of Hantavirus HTNV type;

[0028] Figure 2 This is the result of the intronic primer screening for Hantavirus SEOV type;

[0029] Figure 3 This is the result of peripheral primer screening for Hantavirus HTNV type;

[0030] Figure 4 This is the result of peripheral primer screening for Hantavirus SEOV type;

[0031] Figure 5 Temperature optimization results for Hantavirus HTNV type;

[0032] Figure 6 Temperature optimization results for Hantavirus SEOV type;

[0033] Figure 7 It is the specific test result of Hantavirus HTNV type;

[0034] Figure 8 It is the specific test result of Hantavirus SEOV type;

[0035] Fig. 9 This is the result of the sensitivity test of Hantavirus HTNV type nested MIRA;

[0036] Fig.10 This is the sensitivity test result of common MIRA for Hantavirus HTNV type;

[0037] Fig.11 This is the result of the sensitivity test of Hantavirus SEOV type nested MIRA;

[0038] Fig.12 It is a common MIRA sensitivity test for Hantavirus SEOV type. DETAILED DESCRIPTION

[0039] The technical scheme of the present invention is further described in detail below through examples, but the content of the present invention is not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the materials and reagents used are obtained from commercial channels or prepared according to conventional methods unless otherwise specified;

[0040] Example 1: Design and synthesis of MIRA primer probe

[0041] 1. Primer design and synthesis

[0042] Based on the S fragment sequences of the nucleocapsid protein genes of Hantavirus HTNV and SEOV published by GeneBank, the relatively conservative regions were identified using the biological software Blast to design primer sets and probes for HTNV and SEOV detection, respectively. It should be noted that the design of MIRA primers and probes follows the following principles:

[0043] (1) The length of MIRA primer sequences is within the range of 30-35 bp, following the principle of random distribution of the four bases in the primers to avoid palindromic sequences;

[0044] (2) In the middle of the upstream and downstream primers, a sequence of 46-52 nt in length is used as a fluorescent probe. The probe sequence does not overlap with the specific primer recognition site, and the sequence avoids a palindromic structure and an internal secondary structure. The probe has a total of 4 modification sites: a dSpacer (tetrahydrofuran, THF) is marked in the middle position 30-35 nt away from the 5' end as a recognition site for nuclease exonuclease (any base, no special requirements); a fluorescent group (such as FAM) is marked on the T base at the 5' end of the THF site, and a quenching group (such as BHQ1) is marked on the T base in the 3' direction, and the distance between the two groups is 2-4 nt; THF is about 15 nt away from the 3' end, and a modification group, such as C3-spacer, is marked at the 3' end; the primer probe sequences designed in this embodiment are shown in Tables 1 and 2:

[0045] Table 1 Primer and probe sequences for detection of Hantavirus HTNV

[0046]

[0047] Table 2 Primer and probe sequences for detection of Hantavirus SEOV

[0048]

[0049] 2. Positive plasmid synthesis

[0050] A vector containing a HTNV characteristic sequence whose nucleotide sequence is shown in SEQ ID NO: 11 and a vector containing a SEOV characteristic sequence whose nucleotide sequence is shown in SEQ ID NO: 12 were synthesized by Anhui General Biotechnology;

[0051] The concentration was measured by UV spectrophotometer, and the copy number of the plasmid was calculated according to the length and concentration of each plasmid. The calculation formula is as follows:

[0052]

[0053] Example 2: Establishment and optimization of the nested MIRA detection method for Hantavirus HTNV and SEOV

[0054] In this example, the RNA isothermal rapid amplification kit (fluorescence type) was used to perform the nested MIRA reaction, and the optimal primer set and reaction temperature of the nested MIRA detection method for Hantavirus HTNV type and SEOV type were optimized.

[0055] 1. Screening of the best inner primer set

[0056] (1) Three sets of inner primers were designed for the NP gene of Hantavirus HTNV and SEOV, and they were tested in combination, with 9 combinations for each:

[0057] Hantavirus HTNV type inner primer combinations: HTNV-F1 / HTNV-R1, HTNV-F1 / HTNV-R2, HTNV-F1 / HTNV-R3, HTNV-F2 / HTNV-R1, HTNV-F2 / HTNV-R2, HTNV-F2 / HTNV-R3, HTNV-F3 / HTNV-R1, HTNV-F3 / HTNV-R2, HTNV-F3 / HTNV-R3;

[0058] Hantavirus SEOV type inner primer combination: SEOV-F1 / SEOV-R1, SEOV-F1 / SEOV-R2, SEOV-F1 / SEOV-R3, SEOV-F2 / SEOV-R1, SEOV-F2 / SEOV-R2, SEOV-F2 / SEOV-R3, SEOV-F3 / SEOV-R1, SEOV-F3 / SEOV-R2, SEOV-F3 / SEOV-R3.

[0059] (2) According to the instructions of the RNA Constant Temperature Rapid Amplification Kit (Fluorescence Type), the MIRA reaction system shown in Table 3 was prepared, with a concentration of 1.42×10 7 The plasmid standard with 100 copies / μL was used as a template to test the combination of each primer set and probe.

[0060] Table 3: MIRA reaction system

[0061]

[0062] Note: The probes used are the detection probes corresponding to the Hantavirus HTNV type and the Hantavirus SEOV type;

[0063] (3) Interpretation of results: The results are determined based on the presence or absence of the amplified model. The results of the inner primer screening test are as follows: Figure 1 , Figure 2 As shown, the best internal primer combination for Hantavirus HTNV type is: HTNV-F1 / HTNV-R1; the best internal primer combination for Hantavirus SEOV type is: SEOV-F2 / SEOV-R1.

[0064] 2. Screening of the best peripheral primer set

[0065] (1) Three sets of peripheral primers were designed for the NP gene of Hantavirus HTNV and SEOV, and they were tested in combination, with 9 combinations for each:

[0066] Hantavirus HTNV type peripheral primer combinations: HTNV-WF1 / HTNV-WR1, HTNV-WF1 / HTNV-WR2, HTNV-WF1 / HTNV-WR3, HTNV-WF2 / HTNV-WR1, HTNV-WF2 / HTNV-WR2, HTNV-WF2 / HTNV-WR3, HTNV-WF3 / HTNV-WR1, HTNV-WF3 / HTNV-WR2, HTNV-WF3 / HTNV-WR3;

[0067] Hantavirus SEOV type peripheral primer combinations: SEOV-WF1 / SEOV-WR1, SEOV-WF1 / SEOV-WR2, SEOV-WF1 / SEOV-WR3, SEOV-WF2 / SEOV-WR1, SEOV-WF2 / SEOV-WR2, SEOV-WF2 / SEOV-WR3, SEOV-WF3 / SEOV-WR1, SEOV-WF3 / SEOV-WR2, SEOV-WF3 / SEOV-WR3;

[0068] (2) Based on the best inner primer combination of Hantavirus HTNV and Hantavirus SEOV, the above outer primer combination and the best inner primer combination were subjected to nested MIRA test at a concentration of 1.42×10 7 The plasmid standard with 100 copies / mL was used as a template to screen out the best peripheral primer set; the Nest MIRA reaction system is shown in Table 4:

[0069] Table 4

[0070]

[0071]

[0072] The Nested MIRA test is divided into two steps. The first step is to use peripheral primers to pre-amplify the target sequence. The reaction time is 10 minutes. After the reaction is completed, the first-step product is added to the second-step reaction system, and the target sequence is amplified for the second time by internal primers. During this period, a fluorescence quantification instrument or a convenient fluorescence reader is used to collect signals.

[0073] (3) Interpretation of results: The results are determined based on the presence or absence of the amplified model. Figure 3 , Figure 4 As shown, the best peripheral primer set for Hantavirus HTNV type is: HTNV-WF3 / HTNV-WR1; the best peripheral primer set for Hantavirus SEOV type is: SEOV-WF2 / SEOV-WR1.

[0074] 2. Reaction temperature optimization

[0075] With a concentration of 1.42×10 7 100 copies / mL plasmid standard was used as nucleic acid template, and the nested MIRA reaction system was prepared according to step 1, table 4, and the reaction was carried out under five temperature gradients (35°C, 37°C, 39°C, 41°C, and 43°C). After mixing, the reaction solution was quickly centrifuged and immediately placed in the fluorescence detection equipment for real-time fluorescence quantitative PCR (RT-PCR), the corresponding parameters were set, the fluorescence value of the FAM channel was collected, and the obtained real-time fluorescence data were analyzed.

[0076] Temperature optimization verification results: Figure 5 , 6 As shown, the optimal reaction temperature for Hantavirus HTNV and Hantavirus SEOV is 39°C.

[0077] Example 3: Specificity experiment of the nested MIRA detection method for Hantavirus HTNV and SEOV

[0078] The reaction system and reaction conditions preferred in Example 2 were used to prepare the reaction system and perform the nested MIRA reaction for specificity testing. A total of 9 viruses were used in this example, including Hantavirus HTNV, Hantavirus SEOV, HCoV-229E, fever with thrombocytopenia syndrome virus (SFTSV), Crimean-Congo hemorrhagic fever virus (CCHFV), Coxsackievirus A16 (CVA16), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and mouse norovirus (NV).

[0079] Viral RNA was extracted and nested MIRA amplification reaction was performed using Hantavirus plasmid as a positive control and distilled water as a negative control;

[0080] Test results Figure 7 , Figure 8 As shown, only Hantavirus HTNV and Hantavirus SEOV showed obvious fluorescence curves, while the other 7 viruses and the negative control did not show fluorescence curves, indicating that the Hantavirus HTNV and SEOV nested MIRA detection method established in the present invention has high specificity and no cross-reaction with other pathogens.

[0081] Example 4: Sensitivity experiment of the nested MIRA detection method for Hantavirus HTNV and SEOV

[0082] 1. Preparation of positive template

[0083] The positive standard plasmids of Hantavirus HTNV and Hantavirus SEOV (50 ng / μL) were diluted 10-fold with enzyme-free water to a concentration of 1.42×10 9 copies / mL, 1.42×10 8 copies / mL, 1.42×10 7 copies / mL, 1.42×10 6 copies / mL, 1.42×10 5 copies / mL, 1.42×10 4 copies / mL, 1.42×10 3 copies / mL, 1.42×10 2 A total of 8 copies / mL were used as templates.

[0084] (2) Reaction system preparation

[0085] According to the nested MIRA detection method for Hantavirus HTNV and SEOV established in Example 2, the nested MIRA reaction system was prepared to evaluate the detection sensitivity of Hantavirus HTNV and SEOV. At the same time, a common MIRA reaction system was prepared according to step (1) in Example 2 for comparative study.

[0086] (3) MIRA amplification and result interpretation:

[0087] The amplification reactions of nested MIRA and common MIRA were carried out at 39°C. The reaction time of nested MIRA was 30 min, and that of common MIRA was 20 min. The reaction was terminated and the results were determined based on the amplification curve.

[0088] Test results such as Fig. 9 , Fig.11 As shown in Figure 2, the detection limit of MIRA for Hantavirus HTNV is 1.42×10 4copies / mL, and the detection limit for Hantavirus SEOV was 1.42×10 4 The detection limit of ordinary MIRA is 10 times higher than that of ordinary MIRA, which is 1.42×10 5 copies / mL( Fig.10 , Fig.12 ).

[0089] In summary, the present invention has developed a method for typing and detecting Hantavirus HTNV and SEOV based on the Nest MIRA technology. The technical advantages of the present invention include: (1) Compared with technologies such as RT-qPCR, the operation is simple. It is fast and convenient, and the detection time is greatly shortened. The amplification reaction can be completed in only 30 minutes; (2) This method has strong specificity, and the detection sensitivity can reach 14.2 copies / μL, which is 10 times higher than that of ordinary MIRA; (3) This detection method does not require complex and expensive large equipment, overcomes the limitation of RT-qPCR's dependence on detection equipment, and can be applied to on-site detection.

[0090] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A nested MIRA primer combination for detecting Hantavirus HTNV and SEOV, characterized in that: It includes peripheral primers, internal primers and probes for detecting Hantavirus HTNV type and Hantavirus SEOV type; The peripheral primers for Hantavirus HTNV type are shown in SEQ ID NO: 1 and SEQ ID NO: 2, the inner primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the probe is shown in SEQ ID NO: 5; The peripheral primers for Hantavirus SEOV type are shown in SEQ ID NO:6 and SEQ ID NO:7, the internal primers are shown in SEQ ID NO:8 and SEQ ID NO:9, and the probe is shown in SEQ ID NO:

10.

2. The nested MIRA primer combination for detecting Hantavirus HTNV type and SEOV type according to claim 1, characterized in that: The probe for Hantavirus HTNV type was modified with a FAM fluorescent group at 30 bp from the 5' end, replaced by THF at 33 bp, modified with a BHQ1 group at 34 bp, and the 3' end of the probe was modified with a C3 Spacer group; The probe for Hantavirus SEOV type was modified with a FAM fluorescent group at 32 bp from the 5' end, replaced by THF at 34 bp, modified with a BHQ1 group at 35 bp, and the 3' end of the probe was modified with a C3 Spacer group.

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