Multiplex nucleic acid detection method for eight hemorrhagic fever viruses based on double-probe hybridization and application of multiplex nucleic acid detection method

Abstract

The invention discloses a multiplex nucleic acid detection method for eight hemorrhagic fever viruses based on double-probe hybridization and application of the multiplex nucleic acid detection method. The invention designs and screens eight important virulent hemorrhagic fever viruses (Lassa fever virus, Sudan Ebola virus, Zaire Ebola virus, yellow fever virus, Marburg virus, Maautumn Bo virus, Rift Valley fever virus and Sinnocypress virus) to obtain a set of primer probe set suitable for single-tube multiplex qPCR (quantitative polymerase chain reaction) detection. Conditions such as enzyme reaction liquid, primer probe concentration, THO: PCO ratio and the like used for single-tube multiplex qPCR amplification are explored and optimized, and a sensitive and specific single-tube multiplex qPCR detection method for eight hemorrhagic fever viruses is established. Compared with the prior art, the single-tube multiple qPCR detection method has the advantages that the eight hemorrhagic fever viruses can be detected and identified through one-time amplification reaction, and compared with multiple single detection, the detection efficiency is remarkably improved, and the detection cost is remarkably reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a multiplex nucleic acid detection method for 8 hemorrhagic fever viruses based on dual-probe hybridization and its application. Background Art

[0002] Viral hemorrhagic fever is a general term for a class of viral infectious diseases with symptoms such as fever and hemorrhagic fever caused by infection with various viral pathogens. It includes Marburg virus (MARV), Sin Nombre virus (SNV), Lassa virus (LASV), Sudan Ebola virus (SEBOV), Zaire Ebola virus (ZEBOV), Machupo virus (MACV), Rift Valley fever virus (RVFV), Yellow fever virus (YFV), etc. The above pathogens are all enveloped RNA viruses, prone to mutation. And most of them are highly lethal, seriously endangering human health.

[0003] Marburg virus belongs to the genus Marburgvirus in the family Filoviridae. It is a single-stranded negative-sense RNA virus. The virus particles are long filaments with varying sizes, with diameters ranging from 80 nm to 14,000 nm, and the full-length genome is 19 kb. Ebola virus belongs to the genus Ebolavirus in the family Filoviridae. It is a single-stranded negative-sense RNA virus with a full-length genome of 18.9 kb and can be divided into 4 different serotypes. Among them, Ebola virus Zaire and Ebola virus Sudan can cause severe Ebola hemorrhagic fever. The fatality rate caused by Ebola virus Zaire can reach over 80%, and the fatality rate caused by Ebola virus Sudan reaches over 50%. Sin Nombre virus belongs to the genus Hantavirus in the family Bunyaviridae. It is a single-stranded negative-sense RNA virus. The particles are mostly spherical, with diameters of 80 - 120 nm. Yellow fever virus belongs to the genus Flavivirus in the family Flaviviridae. It is a single-stranded positive-sense RNA virus, generally spherical in shape, with viscerotropism and neurotropism. Machupo virus belongs to the genus Mammarenavirus in the family Arenaviridae. It is a single-stranded negative-sense RNA virus, and the virus particles are spherical and pleomorphic. Rift Valley fever virus belongs to the genus Phlebovirus in the family Phenuiviridae. It is a single-stranded negative-sense RNA virus, generally spherical in shape. Lassa fever virus belongs to the genus Arenavirus in the family Arenaviridae. The viral nucleic acid is negative-strand RNA in two segments, ranging from round to pleomorphic. The virus particles usually contain electron-dense granules and appear sandy red under the electron microscope. The clinical symptoms caused by these eight pathogens are very similar, almost all with fever, bleeding, congestion, and kidney damage as the main clinical manifestations. It is difficult to make a differential diagnosis after virus infection. To screen for the source of infection, multiple tests are required. This brings great difficulties to clinical diagnosis. Currently, there are no commercial detection reagents, and the screening and detection of the above viruses in the laboratory mainly use real-time fluorescence quantitative PCR technology.

[0004] Real-time fluorescence quantitative PCR technology refers to a method in which a Taqman probe labeled with a fluorescent group is added to the PCR reaction system. After completing the thermal cycle of high-temperature denaturation, low-temperature renaturation, and appropriate-temperature extension, the target gene is amplified and hybridized with the probe. Utilizing the exonuclease activity of the polymerase to cleave the Taqman probe complementary to the template DNA, fluorescence signal accumulation is generated to monitor the entire PCR process in real time. Finally, quantitative analysis of the unknown template is carried out through a standard curve. Each fluorescence channel of real-time fluorescence quantitative PCR can only detect one pathogen. Currently, conventional PCR instruments have 4 - 6 fluorescence detection channels. Different fluorescent labels are used for the detection probes of multiple target viruses, enabling the detection of 4 - 6 pathogens in a single reaction tube. However, there are hundreds of common virus species. For specific symptomatic infections, usually 5 - 10 targets need to be screened to identify the pathogen, and conventional qPCR detection is difficult to meet the detection requirements for infectious disease prevention and control. There is an urgent need to establish a higher-throughput nucleic acid detection technology.

[0005] The single-tube multiplex qPCR detection technology combines the TaqMan probe technology and the melting curve technology, which can significantly increase the types of pathogens detected in the same reaction tube. First, multiple pathogen target probes are divided into 3-4 groups, and each group of probes is labeled with a kind of fluorescence. The positive result can be judged which probe group it belongs to through the fluorescence channel selection; then for different probes in the same group, different probe melting temperatures are generated by designing THO / PCO double-labeled probes, and the pathogen probe to which the positive result belongs is determined by analyzing the melting curve temperature. During the amplification process, if there is one or more targets, the corresponding probes are consumed during the probe hydrolysis process, thereby generating amplified fluorescence signals and generating the final melting curve. When judging the results, a two-dimensional method of fluorescence detection channels (FAM, VIC, ROX, CY5) and melting curve Tm value determination is used to determine the detection results. The positive amplification of which group of pathogen nucleic acids is determined by the amplification peaks in different fluorescence channels; furthermore, the melting curves of each sample and the negative control are compared, and the Tm values with reduced characteristic peaks of the melting curves are compared to determine which specific probe is consumed to judge the type of detected positive pathogen, so that 6-10 kinds of pathogens can be detected in a single reaction tube. The principle and process of this technology are as Figure 1 shown, and the main technical difficulties of its core technology mainly lie in designing and screening primer probe sequences without interference with each other, designing THO-PCO probe combinations with specific Tm values, and establishing a reaction system that can sensitively and specifically detect target pathogens. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to establish a sensitive and specific single-tube multiplex qPCR detection method for 8 hemorrhagic fever viruses based on the single-tube multiplex qPCR detection technology.

[0007] To solve the above technical problems, the present invention first provides a set of primer probe groups for detecting 8 hemorrhagic fever viruses.

[0008] The set of primer probe groups for detecting 8 hemorrhagic fever viruses provided by the present invention includes a primer probe group for detecting Lassa fever virus, a primer probe group for detecting Sudan Ebola virus, a primer probe group for detecting Zaire Ebola virus, a primer probe group for detecting yellow fever virus, a primer probe group for detecting Marburg virus, a primer probe group for detecting Machupo virus, a primer probe group for detecting Rift Valley fever virus, and a primer probe group for detecting Sin Nombre virus;

[0009] The primer probe group for detecting Lassa fever virus is composed of an upstream primer of Lassa fever virus, a downstream primer of Lassa fever virus, a THO probe of Lassa fever virus, and a PCO probe of Lassa fever virus;

[0010] The primer-probe set for detecting Sudan ebolavirus consists of an upstream primer for Sudan ebolavirus, a downstream primer for Sudan ebolavirus, a THO probe for Sudan ebolavirus, and a PCO probe for Sudan ebolavirus;

[0011] The primer-probe set for detecting Zaire ebolavirus consists of an upstream primer for Zaire ebolavirus, a downstream primer for Zaire ebolavirus, a THO probe for Zaire ebolavirus, and a PCO probe for Zaire ebolavirus;

[0012] The primer-probe set for detecting yellow fever virus consists of an upstream primer for yellow fever virus, a downstream primer for yellow fever virus, a THO probe for yellow fever virus, and a PCO probe for yellow fever virus;

[0013] The primer-probe set for detecting Marburg virus consists of an upstream primer for Marburg virus, a downstream primer for Marburg virus, a THO probe for Marburg virus, and a PCO probe for Marburg virus;

[0014] The primer-probe set for detecting Machupo virus consists of an upstream primer for Machupo virus, a downstream primer for Machupo virus, a THO probe for Machupo virus, and a PCO probe for Machupo virus;

[0015] The primer-probe set for detecting Rift Valley fever virus consists of an upstream primer for Rift Valley fever virus, a downstream primer for Rift Valley fever virus, a THO probe for Rift Valley fever virus, and a PCO probe for Rift Valley fever virus;

[0016] The primer-probe set for detecting Sin Nombre virus consists of an upstream primer for Sin Nombre virus, a downstream primer for Sin Nombre virus, a THO probe for Sin Nombre virus, and a PCO probe for Sin Nombre virus;

[0017] The upstream primer for Lassa fever virus is the single-stranded DNA molecule shown in Sequence 1;

[0018] The downstream primer for Lassa fever virus is the single-stranded DNA molecule shown in Sequence 2;

[0019] The THO probe for Lassa fever virus is the single-stranded DNA molecule shown in Sequence 3;

[0020] The PCO probe for Lassa fever virus is the single-stranded DNA molecule shown in Sequence 4;

[0021] The upstream primer for Sudan ebolavirus is the single-stranded DNA molecule shown in Sequence 5;

[0022] The downstream primer for Sudan ebolavirus is the single-stranded DNA molecule shown in Sequence 6;

[0023] The THO probe for Sudan ebolavirus is the single-stranded DNA molecule shown in Sequence 7;

[0024] The Sudan Ebola virus PCO probe is a single-stranded DNA molecule shown in Sequence 8;

[0025] The upstream primer of the Zaire Ebola virus is a single-stranded DNA molecule shown in Sequence 5;

[0026] The downstream primer of the Zaire Ebola virus is a single-stranded DNA molecule shown in Sequence 9;

[0027] The Zaire Ebola virus THO probe is a single-stranded DNA molecule shown in Sequence 10;

[0028] The Zaire Ebola virus PCO probe is a single-stranded DNA molecule shown in Sequence 11;

[0029] The upstream primer of the yellow fever virus is a single-stranded DNA molecule shown in Sequence 12;

[0030] The downstream primer of the yellow fever virus is a single-stranded DNA molecule shown in Sequence 13;

[0031] The yellow fever virus THO probe is a single-stranded DNA molecule shown in Sequence 14;

[0032] The yellow fever virus PCO probe is a single-stranded DNA molecule shown in Sequence 15;

[0033] The upstream primer of the Marburg virus is a single-stranded DNA molecule shown in Sequence 16;

[0034] The downstream primer of the Marburg virus is a single-stranded DNA molecule shown in Sequence 17;

[0035] The Marburg virus THO probe is a single-stranded DNA molecule shown in Sequence 18;

[0036] The Marburg virus PCO probe is a single-stranded DNA molecule shown in Sequence 19;

[0037] The upstream primer of the Machupo virus is a single-stranded DNA molecule shown in Sequence 20;

[0038] The downstream primer of the Machupo virus is a single-stranded DNA molecule shown in Sequence 21;

[0039] The Machupo virus THO probe is a single-stranded DNA molecule shown in Sequence 22;

[0040] The Machupo virus PCO probe is a single-stranded DNA molecule shown in Sequence 23;

[0041] The upstream primer of the Rift Valley fever virus is a single-stranded DNA molecule shown in Sequence 24;

[0042] The downstream primer of the Rift Valley fever virus is a single-stranded DNA molecule shown in Sequence 25;

[0043] The THO probe of the Rift Valley fever virus is a single-stranded DNA molecule shown in Sequence 26;

[0044] The PCO probe of the Rift Valley fever virus is a single-stranded DNA molecule shown in Sequence 27;

[0045] The upstream primer of the Sin Nombre virus is a single-stranded DNA molecule shown in Sequence 28;

[0046] The downstream primer of the Sin Nombre virus is a single-stranded DNA molecule shown in Sequence 29;

[0047] The THO probe of the Sin Nombre virus is a single-stranded DNA molecule shown in Sequence 30;

[0048] The PCO probe of the Sin Nombre virus is a single-stranded DNA molecule shown in Sequence 31.

[0049] In the above-mentioned set of primer-probe combinations, the 5'-ends of the THO probes are all labeled with a fluorescent group, and the 3'-ends of the THO probes are all labeled with a quenching group.

[0050] Furthermore, the 5'-ends of the THO probe of the Zaire ebolavirus, the THO probe of the Sudan ebolavirus, and the THO probe of the yellow fever virus are all labeled with fluorescent group A.

[0051] The 5'-ends of the THO probe of the Machupo virus, the THO probe of the Marburg virus, and the THO probe of the Rift Valley fever virus are all labeled with fluorescent group B.

[0052] The 5'-ends of the THO probe of the Lassa fever virus and the THO probe of the Sin Nombre virus are all labeled with fluorescent group C.

[0053] The fluorescent group A, the fluorescent group B, and the fluorescent group C are different fluorescent groups.

[0054] The fluorescent group is selected from any one of the following groups: FAM, 6-FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705, Texas Red.

[0055] The quenching group is selected from any one of the following groups: TAMRA, BHQ1, BHQ2, BHQ3, MGB, Dabcy1.

[0056] Even further, the fluorescent group A is the fluorescent group FAM.

[0057] The fluorophore B is the fluorophore Cy5.

[0058] The fluorophore C is the fluorophore ROX;

[0059] The quenching group is the quenching group BHQ1.

[0060] To solve the above technical problems, the present invention also provides a kit for detecting 8 hemorrhagic fever viruses.

[0061] The kit for detecting 8 hemorrhagic fever viruses provided by the present invention contains the above-mentioned set of primer-probes; the functions of the kit are any one of the following a1)-a3):

[0062] a1) Identifying or assisting in identifying whether the virus to be tested is any one of the 8 hemorrhagic fever viruses;

[0063] a2) Detecting or assisting in detecting whether the sample to be tested is infected with any one or several (including all) of the 8 hemorrhagic fever viruses;

[0064] a3) Distinguishing or assisting in distinguishing the 8 hemorrhagic fever viruses;

[0065] The 8 hemorrhagic fever viruses are Lassa fever virus, Sudan Ebola virus, Zaire Ebola virus, Yellow fever virus, Marburg virus, Machupo virus, Rift Valley fever virus and Sin Nombre virus.

[0066] Furthermore, the kit also includes other reagents for real-time fluorescence quantitative PCR, such as Taq enzyme, buffer solution, etc.

[0067] In some preferred embodiments, the other reagents for real-time fluorescence quantitative PCR are Air-DryableTM 1-step RT Qpcr Mix (product number MDX095) from Meridian BIOSCIENCE.

[0068] Even further, the kit also includes a negative control (such as sterile deionized water).

[0069] To solve the above technical problems, the present invention also provides a new use of the above-mentioned set of primer-probes or the above-mentioned kit.

[0070] The present invention provides the above-mentioned set of primer-probes or the above-mentioned kit in any one of the following b1)-b6):

[0071] b1) Identifying or assisting in identifying whether the virus to be tested is any one of the 8 hemorrhagic fever viruses;

[0072] b2) Detecting or assisting in detecting whether the sample to be tested is infected with any one or several (including all) of the 8 hemorrhagic fever viruses;

[0073] b3) Differentiate or assist in differentiating 8 hemorrhagic fever viruses;

[0074] b4) Prepare a product for identifying or assisting in identifying whether a virus to be tested is any one of the 8 hemorrhagic fever viruses;

[0075] b5) Prepare a product for detecting or assisting in detecting whether a sample to be tested is infected with any one or several (including all) of the 8 hemorrhagic fever viruses;

[0076] b6) Prepare a product for differentiating or assisting in differentiating 8 hemorrhagic fever viruses.

[0077] To solve the above technical problems, the present invention finally provides a method for detecting or assisting in detecting whether a sample to be tested is infected with any one or several (including all) of the 8 hemorrhagic fever viruses.

[0078] The method for detecting or assisting in detecting whether a sample to be tested is infected with any one or several (including all) of the 8 hemorrhagic fever viruses provided by the present invention includes the following steps: extracting the nucleic acid of the sample to be tested, using the nucleic acid of the sample to be tested as a template, and performing real-time fluorescence quantitative PCR with the above-mentioned set of primer-probe groups.

[0079] Further, the method further includes the following steps: judging whether the sample to be tested is infected with any one or several (including all) of the 8 hemorrhagic fever viruses according to the amplification curve generated by the real-time fluorescence quantitative PCR, its Ct value, and the Tm value of the melting curve. The method for judging whether the sample to be tested is infected with the 8 hemorrhagic fever viruses according to the amplification curve generated by the real-time fluorescence quantitative PCR, its Ct value, and the Tm value of the melting curve includes the following steps: first, judging whether the nucleic acid of the sample to be tested is negative amplification or positive amplification according to the amplification curve generated by the real-time fluorescence quantitative PCR and its Ct value (Ct value < 36 is judged as positive amplification); then, performing melting curve analysis on the sample with positive amplification, and judging whether the sample to be tested is infected with any one or several (including all) of the 8 hemorrhagic fever viruses according to the Tm value of the melting curve.

[0080] Still further, the melting curve analysis of the sample with positive amplification includes the following steps: if the signal value of the melting curve peak at a specific Tm value of the sample generating a positive amplification peak is significantly reduced, it is judged that the target virus corresponding to the Tm value of this fluorescence channel is detected as positive.

[0081] Even further, the specific Tm value corresponding to each virus is as follows:

[0082] The specific Tm values corresponding to Sudan Ebola virus, Zaire Ebola virus, and yellow fever virus in the FAM fluorescence channel are 30 - 40 °C, 40 - 50 °C, and 55 - 65 °C respectively.

[0083] The specific Tm values corresponding to Marburg virus, Machupo virus, and Rift Valley fever virus in the Cy5 fluorescence channel are 30 - 40 °C, 40 - 50 °C, and 55 - 65 °C, respectively.

[0084] The specific Tm values corresponding to Lassa fever virus and Sin Nombre virus in the ROX fluorescence channel are 40 - 50 °C and 55 - 65 °C, respectively.

[0085] In the above method, the reaction system of the real-time fluorescence quantitative PCR includes an upstream primer mixture, a downstream primer mixture, a THO probe mixture, and a PCO probe mixture.

[0086] The volume ratio of the upstream primer mixture, the downstream primer mixture, the THO probe mixture, and the PCO probe mixture is 1:1:0.5:1.

[0087] In the upstream primer mixture (composed of water, the upstream primer of yellow fever virus, the upstream primer of Sudan Ebola virus, the upstream primer of Zaire Ebola virus, the upstream primer of Lassa fever virus, the upstream primer of Marburg virus, the upstream primer of Machupo virus, the upstream primer of Rift Valley fever virus, and the upstream primer of Sin Nombre virus), the final concentrations of the upstream primer of yellow fever virus, the upstream primer of Sudan Ebola virus, and the upstream primer of Zaire Ebola virus are all 0.25 μM; the final concentrations of the upstream primer of Lassa fever virus, the upstream primer of Marburg virus, the upstream primer of Machupo virus, the upstream primer of Rift Valley fever virus, and the upstream primer of Sin Nombre virus are all 0.5 μM.

[0088] In the downstream primer mixture (composed of water, the downstream primer of yellow fever virus, the downstream primer of Sudan Ebola virus, the downstream primer of Zaire Ebola virus, the downstream primer of Lassa fever virus, the downstream primer of Marburg virus, the downstream primer of Machupo virus, the downstream primer of Rift Valley fever virus, and the downstream primer of Sin Nombre virus), the final concentrations of the downstream primer of yellow fever virus, the downstream primer of Sudan Ebola virus, and the downstream primer of Zaire Ebola virus are all 0.25 μM; the final concentrations of the downstream primer of Lassa fever virus, the downstream primer of Marburg virus, the downstream primer of Machupo virus, the downstream primer of Rift Valley fever virus, and the downstream primer of Sin Nombre virus are all 0.5 μM.

[0089] In the THO probe mixture (composed of water, yellow fever virus THO probe, Sudan ebolavirus THO probe, Zaire ebolavirus THO probe, Lassa fever virus THO probe, Marburg virus THO probe, Machupo virus THO probe, Rift Valley fever virus THO probe, and Sin Nombre virus THO probe), the final concentrations of the yellow fever virus THO probe, the Sudan ebolavirus THO probe, and the Zaire ebolavirus THO probe are all 0.125 μM; the final concentrations of the Lassa fever virus THO probe, the Marburg virus THO probe, the Machupo virus THO probe, the Rift Valley fever virus THO probe, and the Sin Nombre virus THO probe are all 0.25 μM.

[0090] In the PCO probe mixture (composed of water, yellow fever virus PCO probe, Sudan ebolavirus PCO probe, Zaire ebolavirus PCO probe, Lassa fever virus PCO probe, Marburg virus PCO probe, Machupo virus PCO probe, Rift Valley fever virus PCO probe, and Sin Nombre virus PCO probe), the final concentrations of the yellow fever virus PCO probe, the Sudan ebolavirus PCO probe, and the Zaire ebolavirus PCO probe are all 0.25 μM; the final concentrations of the Lassa fever virus PCO probe, the Marburg virus PCO probe, the Machupo virus PCO probe, the Rift Valley fever virus PCO probe, and the Sin Nombre virus PCO probe are all 0.5 μM.

[0091] In some embodiments, the reaction system of the real-time fluorescence quantitative PCR consists of 5 μl of Air-DryableTM 1-step RT Qpcr Mix (Meridian BIOSCIENCE, MDX095), 1 μl of the upstream primer mixture, 1 μl of the downstream primer mixture, 0.5 μl of the THO probe mixture, 1 μl of the PCO probe mixture, 9.5 μl of sterile deionized water, and 2 μl of the sample to be tested.

[0092] The reaction program of the real-time fluorescence quantitative PCR is as follows: First, 55 °C for 10 min, 95 °C for 3 min; then amplification: 95 °C, 10 s; 60 °C, 45 s, 69 °C, 20 s, for a total of 41 cycles; finally, melting curve analysis: 45 °C, 20 s, 35 °C, 20 s, 25 °C, 1 min; set the heating rate to 68 °C from 25 °C at a rate of 0.05 °C / s, and continuously collect fluorescence.

[0093] Any of the above nucleic acids is RNA.

[0094] Any of the above-mentioned samples to be tested can be various samples carrying the above 8 hemorrhagic fever viruses (one or several or all) or their nucleic acids, including whole blood samples, sputum samples, urine, feces, and vomitus from virus-infected individuals, domestic sewage, surface water, drinking water, etc. from the environment.

[0095] The present invention first selects relatively conserved regions in the gene sequences of 8 target viruses (Marburg virus, Sin Nombre virus, Lassa fever virus, Ebola virus, Zaire ebolavirus, Sudan ebolavirus, Machupo virus, Rift Valley fever virus, Yellow fever virus) as amplification targets, designs amplification primers, and then designs specific probe sequences, namely THO probes, that can recognize various target nucleic acid sequences between the primer pairs. Next, the THO probes of the 8 viruses are grouped, with 2 - 3 THO probes in a group, a total of 3 groups. Each group of THO probes is labeled with FAM, CY5, and ROX fluorescence respectively. Then, for each THO probe, a reverse complementary PCO probe is designed, and by adjusting the length of the PCO probe and the number of bases mismatched with the THO probe, the Tm value of each THO - PCO probe pair is regulated, so that the Tm values of the detection probes for different targets in the same detection channel have appropriate differences, and thus multiple targets can be qualitatively detected in each fluorescence channel. By analyzing the amplification curves and melting curves of different fluorescence channels, the screening and detection of 8 target pathogens in a single reaction tube can be achieved.

[0096] The beneficial effects of the present invention are as follows: The present invention designs and screens a set of primer - probe sets suitable for single - tube multiplex qPCR detection for 8 important virulent hemorrhagic fever viruses (Lassa fever virus, Sudan ebolavirus, Zaire ebolavirus, Yellow fever virus, Marburg virus, Machupo virus, Rift Valley fever virus, and Sin Nombre virus), and explores and optimizes conditions such as the enzyme reaction solution, primer - probe concentration, and THO:PCO ratio used in single - tube multiplex qPCR amplification, thereby establishing a sensitive and specific single - tube multiplex qPCR detection method for 8 hemorrhagic fever viruses. Compared with the prior art, the single - tube multiplex qPCR detection method of the present invention can detect and identify 8 hemorrhagic fever viruses through a single amplification reaction. Compared with multiple single - detection methods, the detection efficiency is significantly improved, and the detection cost is significantly reduced. Description of the Drawings

[0097] Figure 1 It is a schematic diagram of the amplification principle. A: Traditional fluorescence quantitative PCR; B is single - tube multiplex fluorescence quantitative PCR based on the melting curve.

[0098] Figure 2 It is the amplification result diagram of the primer - probe pairs screened for 8 viruses.

[0099] Figure 3Melting curve peaks of THO:PCO probes screened for each virus. A shows the melting curve peaks of three target viruses in the FAM fluorescence channel; B shows the melting curve peaks of three target viruses in the CY5 fluorescence channel; C shows the melting curve peaks of three target viruses in the ROX fluorescence channel.

[0100] Figure 4 Comparison chart of the detection results of two enzyme reagents. A is the FAM channel: SEBOV (10 3 、10 4 、10 5 ); CY5 channel: MARV (10 3 、10 4 、10 5 ); C: ROX channel: SNV (10 3 、10 4 、10 5 ).

[0101] Figure 5 Proportion optimization of primers, THO probes, and PCO probes for single-tube multiplex qPCR detection. A shows the amplification results of system 1; B shows the amplification results of system 2.

[0102] Figure 6 Amplification results of the optimized primers.

[0103] Figure 7 Sensitivity evaluation of the single-tube multiplex qPCR detection method.

[0104] Figure 8 Specificity evaluation of the single-tube multiplex qPCR detection method.

[0105] Figure 9 Virus or pseudovirus sample testing.

[0106] Figure 10 Single-tube multiplex nucleic acid detection of mixed virus samples. A is a mixed sample of Marburg virus and yellow fever virus in tube 1; B is a mixed sample of Rift Valley fever virus and Lassa fever virus in tube 2; C is a mixed sample of Sin Nombre virus and Lassa fever virus in tube 3; D is a mixed sample of Marburg virus, Ebola virus Zaire strain, and Lassa fever virus. Specific implementation mode

[0107] The present invention will be further described in detail below in conjunction with the specific implementation mode. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0108] In the following examples, the experimental methods are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0109] The reagents and their sources involved in the following examples are as follows: nucleic acid extraction kit RNA / DNA MiniKit is a product of Thermo Fisher Scientific (Invditrogen), with the catalog number REF: 12280-050; qPCR amplification reagent TaqMan TM Fast Virus 1-Step Master Mix is a product of Thermo Fisher Scientific (Appliedbiosystems), with the catalog number 4444427; qPCR amplification reagent Air-Dryable TM 1-Step RT-qPCR Mix (4X) is a product of Meridian BIOSCIENCE, with the catalog number MDX095. BL21-DE3 bacteria are products of Beijing Bomed Gene Technology Co., Ltd., with the catalog number BC201-02.

[0110] The fluorescence quantitative PCR instrument in the following examples is a product of BIO-RAD, with the model CFX Opus 96.

[0111] The yellow fever virus 17D vaccine strain in the following examples is described in the literature "Fu Wenchuan, Guo Yinhan, Zhang Lishu, et al. Construction of a recombinant yellow fever virus 17D vaccine expression vector containing the 2A fragment [J]. Chinese Journal of Biotechnology, 2006, (03): 492-498."

[0112] The PSE-380-MS2 plasmid in the following examples is described in the literature "Nian Qinggong, Kang Xiaoping, Zhang Nana, Yang Yinhui, Qin Chengfeng, Deng Yongqiang. Construction and identification of recombinant pseudovirus particles carrying specific gene fragments of St. Louis encephalitis virus. Military Medical Sciences, 2017, 41(3), 194-198."

[0113] Example 1: Design of primers and probes for single-tube multiplex qPCR detection of 8 viruses and optimization of the detection system

[0114] I. Selection of viral target genes

[0115] The target viruses to be detected in the present invention are the following 8 highly pathogenic hemorrhagic fever viruses: Marburg virus (MARV), Sin Nombre virus (SNV), Lassa fever virus (LASV), Ebola virus, Zaire ebolavirus (ZEBOV), Ebola virus, Sudan ebolavirus (SEBOV), Machupo virus (MACV), Rift Valley fever virus (RVFV), and Yellow fever virus (YFV). Through sequence alignment analysis, gene sequences that are conserved within each virus species and specific between different virus species are selected as the qPCR detection target genes for the 8 target viruses to be detected. The specific sequences are shown in Table 1. The target gene sequences of each virus to be detected are commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the PSE-380-MS2 plasmid respectively, and recombinant plasmids containing the target genes of each virus are constructed respectively. The target gene sequences and the concentrations of the synthesized plasmids are shown in Table 1.

[0116] Table 1. Virus target gene sequences

[0117]

[0118]

[0119]

[0120] II. Design, synthesis and screening of primers and probes

[0121] 1. Design and synthesis of primers and probes

[0122] Based on the 8 virus target gene sequences, primers and probes are designed. Forward and reverse primers and THO probes are designed for each virus. To avoid cross-reactions between the primers and probes of different viruses, multiplex PCR primers and probes are designed using the Multiplex Search function of the software Beacon Designer 7.5. The principles for designing primers and probes are as follows: 1) The length of primers and probes is 19 bp - 25 bp; 2) The Tm value of primers is 54 °C - 64 °C; 3) The Tm value of probes is 59 °C - 79 °C; 4) The length of the amplification product is 100 bp - 150 bp; 5) The maximum ΔG of the hairpin structure between each group of primers is 6.0 kcal / mol; 6) The maximum ΔG of the dimer of each group of primers is 10.0 kcal / mol; 7) The difference in Tm values between each group of amplification systems is ≤ 3 °C.

[0123] After the primer and probe sequences are designed, the 8 viruses are divided into the following 3 groups:

[0124] Group 1: Ebola virus, Zaire ebolavirus, Ebola virus, Sudan ebolavirus, and Yellow fever virus.

[0125] Group 2: Machupo virus, Marburg virus, and Rift Valley fever virus.

[0126] Group 3: Lassa fever virus and Sin Nombre virus.

[0127] The 5'-ends of the THO probes for the first group of viruses are all labeled with the fluorescent group FAM.

[0128] The 5'-ends of the THO probes for the second group of viruses are all labeled with the fluorescent group CY5.

[0129] The 5'-ends of the THO probes for the third group of viruses are all labeled with the fluorescent group ROX.

[0130] The 3'-ends of the THO probes for the three groups of viruses are all labeled with the quenching group BHQ1.

[0131] The primers and probes were all synthesized by Shanghai Sangon Biotech Co., Ltd. One to three sets of primer-probe groups were designed for each virus. By detecting the target virus nucleic acid (the recombinant plasmid constructed in step one) diluted in a 10-fold series, the primer-probe group with higher sensitivity was selected. Among them, the primer sequences of Zaire ebolavirus and Sudan ebolavirus are the same to achieve the common coverage and amplification of the two types of Ebola, while the THO probe sequences are different, which can generate different melting curves for typing. The nucleotide sequences of the primers and probes are shown in Table 2.

[0132] Table 2. Primer and probe sequences

[0133]

[0134]

[0135]

[0136] Note: Y represents C or T; M represents A or C; S represents C or G.

[0137] 2. Screening of primer-probe groups by qPCR amplification

[0138] Configure the following reaction system in the qPCR reaction plate: 1 μl of each of the upstream and downstream primers (10 μM), 1 μl of the THO probe (10 μM), 10 μl of sterile deionized water, 2 μl of the serially diluted template nucleic acid (the recombinant plasmid constructed in step one), and 5 μl of TaqMan TM FastVirus 1-Step Master Mix, 20 μl per reaction well. Place the reaction plate in a fluorescence quantitative PCR reaction instrument for PCR amplification. The amplification program is as follows: 55 °C for 10 min, 95 °C for 3 min, and then 41 cycles of amplification: 95 °C, 10 s; 60 °C, 45 s (collect fluorescence). The results were judged according to the amplification curve and the corresponding Ct value.

[0139] The amplification results are as Figure 2As shown, each curve on the amplification graph represents a sample; in the same reaction system, the higher the nucleic acid concentration, the earlier the S-shaped amplification curve appears, and the Ct value is the number of cycles at which a positive signal is generated; for the amplification of the same target gene with different primer pairs, the primer-probe group with higher sensitivity can detect lower nucleic acid concentrations, that is, more amplification curves can be shown in the amplification graph. Select the primer-probe group with high detection sensitivity and high fluorescence intensity of the amplification curve as the primer-probe group for the multiplex detection system. The designed candidate primer-probe sequences and the lowest detectable target concentrations are shown in Table 2. The best primer-probe groups finally screened for each virus are marked in bold and underlined in Table 2.

[0140] III. Design and Screening of PCO Probes

[0141] 1. Design of PCO Probes

[0142] For the THO probes of each pathogen, reverse complementary PCO probes are designed so that the THO probes and PCO probes in the reaction system hybridize and pair during stepwise annealing, and the melting curve temperature (Tm value) is measured. Eight target viruses are divided into 3 groups, and the THO probes of the 3 groups of viruses are labeled with FAM, CY5, and ROX respectively. Since the melting curve Tm values of each THO-PCO probe pair cannot be accurately predicted, multiple PCO probes with incomplete complementary pairing are designed for each THO probe, and the true Tm values of each THO-PCO probe pair are determined by fluorescence quantitative PCR combined with melting curve analysis, and the THO-PCO probe pairs that match the expected Tm values are screened out.

[0143] In order to make the melting curves generated by the virus detection probes in the same group easy to distinguish, by adjusting the length of the PCO probe and setting several mismatched bases on the PCO probe, the Tm value is adjusted, so that the THO-PCO probe pairs of different pathogens in the same fluorescence channel group can form melting curves with different temperatures. The Tm values of the THO-PCO probe pairs of 2-3 viruses in the same group are set at 30-36 °C; 40-46 °C; 50-56 °C; multiple PCO probes are designed for each virus (Table 3), and the Tm values are verified by qPCR experiments, and the PCO probes that match the expected Tm values are screened out.

[0144] Table 3. Virus Grouping and Expected Tm Values

[0145]

[0146] 2. Screening of PCO Probes

[0147] Further, fluorescence quantitative PCR combined with melting curve analysis is used to determine the Tm values of each group of THO-PCO probe pairs, and the THO-PCO probe pairs that match the expectations are screened out for use in the combination and configuration of the multiplex reaction system.

[0148] The specific reaction system for the screening experiment of eight virus PCO probes is as follows: 1 μl of each of the upstream and downstream primers (10 μmol / L), 1 μl of the THO probe (10 μmol / L), 2 μl of the PCO probe (10 μmol / L), 12 μl of sterile deionized water, and 5 μl of TaqMan TM Fast Virus 1-Step Master Mix. Each reaction system totals 20 μl. The amplification program is as follows: 55 °C for 10 min, 95 °C for 3 min, followed by amplification: 95 °C for 10 s; 60 °C for 45 s, 69 °C for 20 s, for a total of 41 cycles; finally, melting curve analysis is performed: 45 °C for 20 s, 35 °C for 20 s, 25 °C for 1 min; heating from 25 °C to 68 °C at a rate of 0.05 °C / s, continuously collecting fluorescence for melting curve analysis to obtain the Tm value of the melting curve formed by the THO-PCO probe pair in the qPCR reaction system without an amplification template for each virus reaction system.

[0149] The Tm values of each THO-PCO probe pair are shown in Table 4. For some viruses, the Tm values generated by multiple PCO probes meet the expectations. According to the melting curve peaks they produce, the PCO probe with a high fluorescence intensity of the melting curve is selected as the best PCO probe. The best PCO probes corresponding to each virus in each channel are shown in bold and underlined in Table 4, and are intended to be combined with the primer-probe sets screened out in Table 2 to establish a single-tube multiplex nucleic acid detection method. The melting curve graphs of each THO-PCO probe pair that meet the expectations are shown in Figure 3 .

[0150] Table 4. PCO probe sequences and actually measured Tm values

[0151]

[0152]

[0153]

[0154] IV. Optimization of the single-tube multiplex qPCR detection reaction system

[0155] In the single-tube multiplex qPCR detection method established by the present invention, each reaction system contains upstream and downstream primers, THO probes, PCO probes, enzyme reaction solutions, and sterile deionized water. Since enzyme reaction solutions from different manufacturers and different primer-probe usage concentrations will have a significant impact on the detection effect, conditions such as enzyme reaction solutions from different manufacturers, primer-probe concentrations, and THO:PCO ratios were explored and optimized respectively, so as to establish a sensitive and specific single-tube 8-target gene amplification detection technology.

[0156] 1. Selection of amplification reagents

[0157] The enzyme reaction solution reagents produced by Thermo Fisher Scientific (ABI) (TaqMan TM Fast Virus 1-Step MasterMix, hereinafter referred to as ABI reagent) and the enzyme reaction solution reagents produced by Meridian BIOSCIENCE (Air-Dryable TM 1-step RT Qpcr Mix (4X), hereinafter referred to as Meridian reagent) were respectively selected for multiplex system detection. Using Marburg virus, Sudan Ebola virus, and Sin Nombre virus as detection targets, the enzyme reaction solution with better detection effect was screened by comparing the Ct values of the detection results, the fluorescence intensities of the amplification curves and melting curves.

[0158] 1) The upstream primers, downstream primers, THO probes, and PCO probes of 8 target viruses were respectively mixed to prepare an upstream primer mixture (the solvent is water, and the concentration of each virus primer is 1 μM), a downstream primer mixture (the solvent is water, and the concentration of each virus primer is 1 μM), a THO probe mixture (the solvent is water, and the concentration of each virus THO probe is 1 μM), and a PCO probe mixture (the solvent is water, and the concentration of each virus PCO probe is 1 μM).

[0159] 2) Use ABI reagent to prepare the reaction system. The reaction system consists of 1 μl of upstream primer mixture, 1 μl of downstream primer mixture, 1 μl of THO probe mixture, 2 μl of PCO probe mixture, 8 μl of sterile deionized water, 2 μl of target gene plasmid, and 5 μl of TaqMan TM Fast Virus 1-Step MasterMix. At the same time, sterile deionized water was used as the negative control group.

[0160] Use Meridian reagent to prepare the reaction system. The reaction system consists of 1 μl of upstream primer mixture, 1 μl of each downstream primer mixture, 1 μl of THO probe mixture, 2 μl of PCO probe mixture, 8 μl of sterile deionized water, 2 μl of target gene plasmid, and 5 μl of Air-Dryable TM 1-step RT Qpcr Mix (4X). At the same time, sterile deionized water was used as the negative control group.

[0161] 3) Perform real-time fluorescence quantitative PCR on each reaction system. The fluorescence quantitative PCR amplification program is as follows: 55°C for 10 min, 95°C for 3 min, and then perform amplification: 95°C for 10 s; 60°C for 45 s, 69°C for 20 s, a total of 41 cycles; finally, perform melting curve analysis: 45°C for 20 s, 35°C for 20 s, 25°C for 1 min; set the temperature to rise from 25°C to 68°C at a rate of 0.05°C / s, and continuously collect fluorescence.

[0162] 4) Result judgment: First, based on the amplification curve and its Ct value presented in the real-time fluorescence quantitative PCR experiment results, judge the positive and negative amplification of the template nucleic acid (Ct value < 36 is judged as positive); then perform melting curve analysis on the positive samples, and determine the pathogen type according to the Tm value of the melting curve. Since the THO-PCO probe in the negative system is complementary and paired, the THO probe does not produce a fluorescence signal and can produce a typical melting curve peak at a specific temperature (Tm); if positive amplification is achieved in the reaction system, the THO probe in the reaction system is cleaved to produce a fluorescence signal (amplification curve), but the formed THO-PCO probe pair will decrease, and the melting curve peak signal generated at a specific temperature (Tm) will decrease. Therefore, the positive melting curve judgment method in this technical method is as follows: Taking the melting curve peak of the negative control sample as a reference, compare the melting curve changes of the sample with a positive amplification peak in the corresponding fluorescence channel. If the melting curve peak signal value of the sample with a positive amplification peak is significantly reduced at a specific Tm value (see Table 3), that is, a positive melting curve peak appears, it is judged that the target virus detected at the Tm value of this fluorescence channel is positive. Compare the Ct values, amplification curves, and fluorescence intensities of the melting curves of the detection results of the two enzyme reaction solutions. The enzyme reaction solution with a lower Ct value, a higher fluorescence intensity of the amplification curve, and a more significant reduction in the melting curve signal of the positive sample at a specific Tm value is considered to have a better detection effect.

[0163] The amplification results of detecting SEBOV in the FAM fluorescence channel, MARV in the CY5 fluorescence channel, and SNV in the ROX channel by configuring a multiplex detection system with ABI reagents and Meridian reagents respectively are as Figure 4 shown. The results show that for the viral nucleic acid in the ROX channel, both reagents can perform positive amplification and show similar amplification curves and melting curves. For the amplification of SEBOV in the FAM channel, whether it is the amplification curve or the melting curve, Meridian reagents show better amplification effects, higher sensitivity, and more obvious positive melting curves; for the amplification of MARV in the CY5 channel, although the amplification curves are similar, the fluorescence positive results of the melting curves generated by Meridian reagents are more obvious, indicating that Meridian reagents are more suitable for multiplex nucleic acid amplification detection. Therefore, in subsequent experiments, Meridian reagents were selected to study the single-tube multiplex nucleic acid detection method.

[0164] 2. Optimization of primer and probe ratios

[0165] Mix the forward primers, reverse primers, THO probes, and PCO probes of 8 target viruses respectively to prepare a forward primer mixture (solvent is water, and the concentration of each virus primer is 1 μM), a reverse primer mixture (solvent is water, and the concentration of each virus primer is 1 μM), a THO probe mixture (solvent is water, and the concentration of each virus THO probe is 1 μM), and a PCO probe mixture (solvent is water, and the concentration of each virus PCO probe is 1 μM). Use Meridian reagent to prepare a detection system for detecting the nucleic acids of 8 hemorrhagic fever viruses (the specific components are shown in Table 5). Using SNV and MARV as templates, compare the detection efficiencies of two ratios of primer:THO probe:PCO probe of 1:1:2 (System 1) and 1:0.5:1 (System 2) to screen out the optimal primer-probe ratio. The reaction amplification program and the result judgment method are the same as in Step 1.

[0166] Table 5. Optimization of primer and THO probe ratios

[0167]

[0168]

[0169] Using SNV and MARV as templates, compare the detection efficiencies of two ratios of primer:THO probe:PCO probe of 1:1:2 (System 1) and 1:0.5:1 (System 2). The results are as Figure 5 shown. The results show that positive amplification curves and positive melting curve peaks appear in both detection systems. Among them, the positive melting curve peak of System 2 is more obvious. Therefore, the ratio of primer:THO probe:PCO probe of 1:0.5:1 is selected as the best primer-probe ratio for the multiplex detection system.

[0170] 3. Optimization of primer and probe concentrations

[0171] Select 5 different concentrations of primer mixtures and THO probe mixtures, namely 4 μM, 2 μM, 1 μM, 0.5 μM, and 0.25 μM, to prepare 8-plex reaction systems respectively, and amplify 8 target viruses respectively. Compare the detection efficiencies to screen out the most suitable primer-probe concentrations for each virus. The primer-probe concentrations and the composition of the reaction systems of each system are shown in Table 6. The reaction amplification program and the result judgment method are the same as in Step 1.

[0172] Compare the Ct values of the nucleic acid detection results of each virus at different concentrations and the changes in the melting curves. Primer-probe concentrations with lower Ct values, higher fluorescence intensities of the amplification curves, and more significant positive melting curve peaks at the specific Tm values of positive samples are considered to have better detection effects.

[0173] Table 6. Four reaction systems for optimizing primer concentration

[0174]

[0175] The results are shown in Table 7. The results indicate that most viruses can be amplified in different primer-probe concentration systems, but there are significant differences in the amplified Ct values and the peak differences of the melting curves. The primer-probe concentration with the smallest amplified Ct value and the most significant positive melting curve peak was selected as the optimized primer-probe concentration for the single-tube multiplex nucleic acid detection of 8 hemorrhagic fever viruses. The optimal primer-probes for SEBOV, ZEBOV, and YFV in the FAM fluorescence channel were 0.5 μM, and the optimal primer concentrations for viruses in the CY5 channel and ROX channel were 1 μM. The amplification curves and melting curves generated by the best primer-probe concentration selected for each virus are as Figure 6 shown.

[0176] Table 7. Optimization of primer-probe concentration

[0177]

[0178] Example 2. Sensitivity evaluation of the single-tube multiplex qPCR detection method for 8 viruses

[0179] Experimental method: Using the optimal primer-probe concentration and primer-probe ratio for each virus obtained in Example 1, a single-tube multiplex nucleic acid detection system for 8 important hemorrhagic fever viruses was configured. The composition of the single-tube multiplex nucleic acid detection system is shown in Table 8.

[0180] The recombinant plasmids of MARV, SNV, LASV, SEBOV, ZEBOV, MACV, RVFV, and YFV prepared in Example 1 were serially diluted 10-fold as templates, and at the same time, Nuclease-free H2O was used as a template as a negative control to determine the lowest target nucleic acid concentration that this method can detect.

[0181] The amplification conditions and positive judgment criteria were the same as those in Example 1.

[0182] Table 8. Composition of the single-tube multiplex nucleic acid detection system

[0183]

[0184]

[0185] The results are as Figure 7As shown in Table 9. The results show that the lowest detectable copy number of most viruses is 1 - 100 copies / ul, and only the lowest detection concentrations of Rift Valley fever virus and Machupo virus are higher than 100 copies / ul. This indicates that the 8 - multiplex hemorrhagic fever virus nucleic acid detection system can sensitively detect the nucleic acids of 8 target hemorrhagic fever viruses.

[0186] Meanwhile, when detecting each virus, positive signals of the target virus only appeared in the detection wells corresponding to the virus, and no positive signals of other viruses appeared, indicating that the detection system has good specificity and there is no cross - reaction among the 8 viruses.

[0187] Table 9. Results of sensitivity detection

[0188]

[0189] Example 3. Specificity evaluation of the single - tube multiplex qPCR detection method for 8 viruses

[0190] Test samples: Tick - borne encephalitis virus nucleic acid, Japanese encephalitis virus nucleic acid, Chikungunya virus nucleic acid, Respiratory syncytial virus nucleic acid, SARS - CoV - 2 nucleic acid, Enterovirus EV71 nucleic acid, Severe fever with thrombocytopenia syndrome virus nucleic acid, H1N1 influenza virus nucleic acid, Adenovirus nucleic acid, Dengue virus nucleic acid, Nucleic acid of normal human throat swab.

[0191] Tick - borne encephalitis virus is recorded in the literature "Optimization of eukaryotic expression of the extracellular region of the envelope glycoprotein of tick - borne encephalitis virus and its evaluation in serological detection, Huo Naifan, Kang Xiaoping, Hu Yi, Li Yuchang, Li Jing, Zhang Yu, Ran Xin, Jia Jia, Cao Xuefeng, Yang Yinhui, Biotechnology Bulletin, Vol.27 No.3 May, 2016, 38 - 41".

[0192] Adenovirus (ADV), SARS - CoV - 2, Enterovirus EV71, Respiratory syncytial virus, Yellow fever virus YF 17D (YF), Japanese encephalitis virus (JEV), Influenza virus H1N1 subtype and Dengue virus are all recorded in "Tang Y, Wang Y, Li Y, Zhao H, Zhang S, Zhang Y, Li J, Chen Y, Wu X, Qin C, Jiang T, Kang X. An integrated rapid nucleic acid detection assay based on recombinant polymerase amplification for SARS - CoV - 2. Virol Sin. 2022 Feb; 37(1):138 - 141."

[0193] Chikungunya virus is recorded in the literature "Fan Min, Tian Mingyao, Zhao Quan, et al. Establishment of a gene chip for the detection of Chikungunya virus and Sindbis virus [J]. Acta Veterinaria Sinica, 2012, 32(10): 1493-1497."

[0194] Severe fever with thrombocytopenia syndrome virus is recorded in the literature "Wang Chunfang, Luo Zhenghan, Wang Yuhe, et al. Establishment of a method for rapid detection of severe fever with thrombocytopenia syndrome virus by enzyme-catalyzed recombinant isothermal amplification real-time fluorescence method [J]. Chinese Journal of Hygienic Insecticides & Equipments, 2024, 30(06): 554-558. DOI: 10.19821 / j.1671-2781.2024.06.013."

[0195] Experimental method: Using the test sample as a template, fluorescence quantitative PCR was performed using the single-tube multiplex nucleic acid detection system shown in Table 8. The specific amplification conditions and result judgment were the same as in Example 1. At the same time, nucleic acids of RVFV, YFV, and SEBOV in the CY5, FAM, and ROX channels diluted 10 -4 times were used as positive controls.

[0196] The results are as Figure 8 shown. The results show that no positive signals appeared in the nucleic acids of 11 unrelated viruses, while the positive controls amplified well, indicating that the detection system has good specificity and no cross-reaction.

[0197] Example 4. Sample testing of the single-tube multiplex qPCR detection method for 8 viruses

[0198] I. Virus culture or pseudovirus testing

[0199] Since the 8 target viruses of the present invention are all pathogens related to severe hemorrhagic fever, and there is only the yellow fever virus 17D vaccine strain in China, and the other viruses are all rare pathogens, there are no real viruses and real clinical samples for testing. Therefore, for the 7 rare pathogens other than the yellow fever virus, pseudoviruses containing 7 target genes were prepared in the present invention. Nucleic acids of the yellow fever virus 17D culture and the other 7 hemorrhagic fever-related pseudoviruses were used as test samples. After nucleic acid extraction, amplification was performed to determine that the single-tube multiplex qPCR detection method for 8 viruses established in the present invention can detect nucleic acids in the samples. The specific steps are as follows:

[0200] 1. Preparation of pseudoviruses

[0201] The 7 recombinant plasmids containing viral target genes other than yellow fever virus in Example 1 were separately transformed into BL21-DE3 bacteria, and positive clones were picked; the positive clone bacteria were separately transferred to 10 mL of LB medium with ampicillin resistance and cultured with shaking at 37 °C and 180 rpm for 4 - 6 h until the OD value of the bacterial solution was 0.6 - 0.8; IPTG with a final concentration of 1 mM was added, and induction expression was carried out at 16 °C and 180 rpm for 16 h; the bacterial solution was centrifuged at 9000×g for 10 min, and the bacterial cell precipitate was collected; ultrasonication was carried out on ice, and the ultrasonic product was centrifuged at 9000 rpm for 30 min, and the supernatant was collected into a 15 mL centrifuge tube. The supernatant contained the pseudovirus induced and expressed.

[0202] 2. Preparation of yellow fever virus culture

[0203] 1) Passage BHK cells into a T25 cell culture flask and culture at 37 °C under 5% CO2 conditions. The next day, wait until the cells are about 80 - 90% confluent for the experiment.

[0204] 2) Discard the medium in the T25 cell culture flask, and then add 5 mL of maintenance medium containing 2% FBS to the flask.

[0205] 3) Add 100 μL of yellow fever virus 17D to the T25 cell culture flask in the previous step.

[0206] 4) Culture at 37 °C under 5% CO2 conditions.

[0207] 5) Observe daily, and terminate the culture when about 70% of the cells show CPE (cytopathic effect).

[0208] 6) Place the T25 cell culture flask in an ultra-low temperature refrigerator and freeze-thaw 3 times.

[0209] 7) Transfer the liquid in the T25 cell culture flask to a 15 mL centrifuge tube.

[0210] 8) Centrifuge at 4000 rpm for 10 min, take the supernatant (i.e., yellow fever virus culture) and store it in an ultra-low temperature refrigerator at -80 °C for later use.

[0211] 3. Preparation of test samples

[0212] Separate nucleic acid extraction was carried out on the yellow fever virus culture and 7 pseudoviruses as test samples, and each virus was gradient diluted at 10 -5 、10 -6 to prepare 2 samples with different dilution degrees.

[0213] 4. Fluorescent quantitative PCR

[0214] Using the test sample as a template, fluorescence quantitative PCR was performed using the single-tube multiplex nucleic acid detection system shown in Table 8. The specific amplification conditions and result judgment were the same as in Example 1.

[0215] The results were as Figure 9 shown in and Table 10, and the results showed that: among the 16 test samples of 8 viruses, positive could be correctly detected, indicating that the single-tube multiplex nucleic acid detection method established by the present invention can correctly detect the nucleic acid in the sample.

[0216] Table 10. Sample test results

[0217]

[0218]

[0219] II. Mixed sample test

[0220] Prepare the following mixed samples:

[0221] Mixed sample 1: Equal volume mixture of Marburg virus supernatant dilution (10 3 copies / ul) and yellow fever virus culture.

[0222] Mixed sample 2: Equal volume mixture of Rift Valley fever virus supernatant dilution (10 3 copies / ul) and Sin Nombre virus supernatant dilution (10 3 copies / ul concentration).

[0223] Mixed sample 3: Equal volume mixture of Rift Valley fever virus supernatant dilution (10 3 copies / ul) and Marburg virus supernatant dilution (10 3 copies / ul).

[0224] Mixed sample 4: Equal volume mixture of Marburg virus supernatant dilution (10 -6 concentration), Ebola virus Zaire strain supernatant dilution (10 3 copies / ul) and Lassa fever virus supernatant dilution (10 3 copies / ul).

[0225] The pathogens contained in each mixed sample are shown in Table 11.

[0226] Table 11. Viruses contained in the mixed samples

[0227] Number Contained virus 1 Marburg virus, Yellow fever virus 2 Rift Valley fever virus, Sin Nombre virus 3 Marburg virus, Rift Valley fever 4 Marburg virus, Ebola virus Zaire strain and Lassa fever virus

[0228] Using the mixed samples 1, 2, 3, and 4 as templates respectively, fluorescence quantitative PCR was performed using the single-tube multiplex nucleic acid detection system shown in Table 8. The specific amplification conditions and result judgment were the same as those in Example 1.

[0229] The results are as Figure 10 shown in Table 12, and the results show that all the mixed samples accurately detected the contained viruses, indicating that the single-tube multiplex qPCR detection method for 8 viruses established by the present invention can correctly detect the target virus mixed infection.

[0230] Table 12. Detection results of mixed virus samples

[0231]

[0232] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made with conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A set of primer probe groups for detecting 8 hemorrhagic fever viruses, the primer probe groups comprising a primer probe group for detecting Lassa fever virus, a primer probe group for detecting Sudan Ebola virus, a primer probe group for detecting Zaire Ebola virus, a primer probe group for detecting yellow fever virus, a primer probe group for detecting Marburg virus, a primer probe group for detecting Machupo virus, a primer probe group for detecting Rift Valley fever virus, and a primer probe group for detecting Sin Nombre virus; The primer probe set for detecting Lassa fever virus consists of a Lassa fever virus upstream primer, a Lassa fever virus downstream primer, a Lassa fever virus THO probe and a Lassa fever virus PCO probe; The primer probe set for detecting Sudan Ebola virus consists of Sudan Ebola virus upstream primer, Sudan Ebola virus downstream primer, Sudan Ebola virus THO probe and Sudan Ebola virus PCO probe; The primer probe set for detecting Zaire Ebola virus consists of a Zaire Ebola virus upstream primer, a Zaire Ebola virus downstream primer, a Zaire Ebola virus THO probe and a Zaire Ebola virus PCO probe; The primer probe set for detecting yellow fever virus consists of a yellow fever virus upstream primer, a yellow fever virus downstream primer, a yellow fever virus THO probe and a yellow fever virus PCO probe; The primer probe set for detecting Marburg virus consists of a Marburg virus upstream primer, a Marburg virus downstream primer, a Marburg virus THO probe and a Marburg virus PCO probe; The primer probe set for detecting Machupo virus consists of Machupo virus upstream primer, Machupo virus downstream primer, Machupo virus THO probe and Machupo virus PCO probe; The primer probe set for detecting Rift Valley fever virus consists of a Rift Valley fever virus upstream primer, a Rift Valley fever virus downstream primer, a Rift Valley fever virus THO probe and a Rift Valley fever virus PCO probe; The primer probe set for detecting Sin Nombre virus consists of a Sin Nombre virus upstream primer, a Sin Nombre virus downstream primer, a Sin Nombre virus THO probe and a Sin Nombre virus PCO probe; The Lassa fever virus upstream primer is a single-stranded DNA molecule shown in sequence 1; The Lassa fever virus downstream primer is a single-stranded DNA molecule shown in sequence 2; The Lassa fever virus THO probe is a single-stranded DNA molecule shown in sequence 3; The Lassa fever virus PCO probe is a single-stranded DNA molecule shown in sequence 4; The Sudan Ebola virus upstream primer is a single-stranded DNA molecule shown in sequence 5; The Sudan Ebola virus downstream primer is a single-stranded DNA molecule shown in sequence 6; The Sudan Ebola virus THO probe is a single-stranded DNA molecule shown in sequence 7; The Sudan Ebola virus PCO probe is a single-stranded DNA molecule shown in sequence 8; The Zaire Ebola virus upstream primer is a single-stranded DNA molecule shown in sequence 5; The Zaire Ebola virus downstream primer is a single-stranded DNA molecule shown in sequence 9; The Zaire Ebola virus THO probe is a single-stranded DNA molecule shown in sequence 10; The Zaire Ebola virus PCO probe is a single-stranded DNA molecule shown in sequence 11; The yellow fever virus upstream primer is a single-stranded DNA molecule shown in sequence 12; The yellow fever virus downstream primer is a single-stranded DNA molecule shown in sequence 13; The yellow fever virus THO probe is a single-stranded DNA molecule shown in sequence 14; The yellow fever virus PCO probe is a single-stranded DNA molecule shown in sequence 15; The Marburg virus upstream primer is a single-stranded DNA molecule shown in sequence 16; The Marburg virus downstream primer is a single-stranded DNA molecule shown in sequence 17; The Marburg virus THO probe is a single-stranded DNA molecule shown in sequence 18; The Marburg virus PCO probe is a single-stranded DNA molecule shown in sequence 19; The Machupo virus upstream primer is a single-stranded DNA molecule shown in sequence 20; The Machupo virus downstream primer is a single-stranded DNA molecule shown in sequence 21; The Machupo virus THO probe is a single-stranded DNA molecule shown in sequence 22; The Machupo virus PCO probe is a single-stranded DNA molecule shown in sequence 23; The Rift Valley fever virus upstream primer is a single-stranded DNA molecule shown in sequence 24; The Rift Valley fever virus downstream primer is a single-stranded DNA molecule shown in sequence 25; The Rift Valley fever virus THO probe is a single-stranded DNA molecule shown in sequence 26; The Rift Valley fever virus PCO probe is a single-stranded DNA molecule shown in sequence 27; The Sin Nombre virus upstream primer is a single-stranded DNA molecule shown in sequence 28; The Sin Nombre virus downstream primer is a single-stranded DNA molecule shown in sequence 29; The Sin Nombre virus THO probe is a single-stranded DNA molecule shown in sequence 30; The Sin Nombre virus PCO probe is a single-stranded DNA molecule shown in sequence 31.

2. The primer probe set according to claim 1, characterized in that: The 5' ends of the THO probes are all labeled with a fluorescent group, and the 3' ends of the THO probes are all labeled with a quenching group.

3. The primer probe set according to claim 1 or 2, characterized in that: The 5' ends of the Zaire Ebola virus THO probe, the Sudan Ebola virus THO probe and the yellow fever virus THO probe are all labeled with fluorescent group A; the 5' ends of the Machupo virus THO probe, the Marburg virus THO probe and the Rift Valley fever virus THO probe are all labeled with fluorescent group B; the 5' ends of the Lassa fever virus THO probe and the Sin Nombre virus THO probe are all labeled with fluorescent group C; The fluorescent group A, the fluorescent group B and the fluorescent group C are different fluorescent groups.

4. The primer probe set according to claim 1, characterized in that: The fluorescent group A is a fluorescent group FAM; The fluorescent group B is the fluorescent group Cy5; The fluorescent group C is the fluorescent group ROX; The quenching group is the quenching group BHQ1.

5. A kit for detecting eight hemorrhagic fever viruses, the kit comprising a primer probe set according to any one of claims 1 to 4; the function of the kit is any one of the following a1) to a3): a1) Identify or assist in identifying whether the virus to be tested is any of the eight hemorrhagic fever viruses; a2) Detect or assist in detecting whether the sample to be tested is infected with any one or more of the eight hemorrhagic fever viruses; a3) Differentiate or assist in differentiating 8 types of hemorrhagic fever viruses; The eight hemorrhagic fever viruses are Lassa fever virus, Sudan Ebola virus, Zaire Ebola virus, yellow fever virus, Marburg virus, Machupo virus, Rift Valley fever virus and Sin Nombre virus.

6. The kit according to claim 5, characterized in that: The kit also includes other reagents for performing real-time fluorescence quantitative PCR.

7. The primer probe set according to any one of claims 1 to 4 or the kit according to claim 5 or 6 is any one of the following b1) to b6): b1) Identify or assist in identifying whether the virus to be tested is any of the eight hemorrhagic fever viruses; b2) Detect or assist in detecting whether the sample to be tested is infected with any one or more of the eight hemorrhagic fever viruses; b3) Differentiate or assist in differentiating 8 types of hemorrhagic fever viruses; b4) preparing products for identification or auxiliary identification of whether the virus to be tested is any of the eight hemorrhagic fever viruses; b5) Prepare products for detecting or assisting in detecting whether the sample to be tested is infected with any one or more of the eight hemorrhagic fever viruses; b6) Preparation of products that differentiate or assist in differentiating eight hemorrhagic fever viruses; The eight hemorrhagic fever viruses are Lassa fever virus, Sudan Ebola virus, Zaire Ebola virus, yellow fever virus, Marburg virus, Machupo virus, Rift Valley fever virus and Sin Nombre virus.

8. A method for detecting or assisting in detecting whether a sample to be tested is infected with any one or more of eight hemorrhagic fever viruses, comprising the following steps: extracting nucleic acid from the sample to be tested, using the nucleic acid of the sample to be tested as a template, and performing real-time fluorescence quantitative PCR using the primer probe set described in any one of claims 1 to 4; The eight hemorrhagic fever viruses are Lassa fever virus, Sudan Ebola virus, Zaire Ebola virus, yellow fever virus, Marburg virus, Machupo virus, Rift Valley fever virus and Sin Nombre virus.

9. The method according to claim 8, characterized in that The method further comprises the following steps: judging whether the sample to be tested is infected with any one or several of the eight hemorrhagic fever viruses according to the amplification curve and its Ct value generated by the real-time fluorescence quantitative PCR and the Tm value of the melting curve.

10. The method according to claim 8 or 9, characterized in that: The real-time fluorescence quantitative PCR reaction system includes an upstream primer mixture, a downstream primer mixture, a THO probe mixture and a PCO probe mixture; Or, the volume ratio of the upstream primer mixture, the downstream primer mixture, the THO probe mixture and the PCO probe mixture is 1:1:0.5:1; In the upstream primer mixture, the final concentrations of the yellow fever virus upstream primer, the Sudan Ebola virus upstream primer, and the Zaire Ebola virus upstream primer are all 0.25 μM; the final concentrations of the Lassa fever virus upstream primer, the Marburg virus upstream primer, the Machupo virus upstream primer, the Rift Valley fever virus upstream primer, and the Sin Nombre virus upstream primer are all 0.5 μM; In the downstream primer mixture, the final concentrations of the yellow fever virus downstream primer, the Sudan Ebola virus downstream primer, and the Zaire Ebola virus downstream primer are all 0.25 μM; the final concentrations of the Lassa fever virus downstream primer, the Marburg virus downstream primer, the Machupo virus downstream primer, the Rift Valley fever virus downstream primer, and the Sin Nombre virus downstream primer are all 0.5 μM; In the THO probe mixture, the final concentrations of the yellow fever virus THO probe, the Sudan Ebola virus THO probe, and the Zaire Ebola virus THO probe are all 0.125 μM; the final concentrations of the Lassa fever virus THO probe, the Marburg virus THO probe, the Machupo virus THO probe, the Rift Valley fever virus THO probe, and the Sin Nombre virus THO probe are all 0.25 μM; In the PCO probe mixture, the final concentrations of the yellow fever virus PCO probe, the Sudan Ebola virus PCO probe and the Zaire Ebola virus PCO probe are all 0.25 μM; the final concentrations of the Lassa fever virus PCO probe, the Marburg virus PCO probe, the Machupo virus PCO probe, the Rift Valley fever virus PCO probe and the Sin Nombre virus PCO probe are all 0.5 μM.

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