Rapid detection kit for multiple pathogens of hemorrhagic fever

Abstract

The invention relates to the field of virus detection, in particular to a kit for rapidly detecting multiple pathogens of hemorrhagic fever, which comprises a dengue virus upstream primer, a dengue virus downstream primer and a probe for detecting the dengue virus. The kit comprises a chikungunya virus upstream primer, a chikungunya virus downstream primer and a probe for detecting the chikungunya virus, a Zika virus upstream primer, a Zika virus downstream primer and a probe for detecting the Zika virus; the kit comprises a yellow fever virus upstream primer, a yellow fever virus downstream primer and a probe for detecting the yellow fever virus. The kit provided by the invention can quickly and accurately detect and distinguish the hemorrhagic fever pathogen, and the actual detection time is only 24-35 minutes, which is far lower than that of the kit in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of pathogen detection, and particularly to a rapid detection kit for multiple pathogens of hemorrhagic fever. Background Art

[0002] Viral hemorrhagic fevers (VHFs) are a group of natural zoonotic diseases caused by arboviruses, with fever, hemorrhage (including bleeding tendency and vascular damage), and shock as the main clinical features. In tropical and subtropical regions, arboviruses have always been a huge health threat.

[0003] Dengue virus (DENV) is one of the arboviruses that cause viral hemorrhagic fever, mainly transmitted by Aedes mosquito bites. Approximately 390 million people are infected with dengue fever worldwide every year, and about 96 million people are ill, affecting more than 120 countries, with the most serious cases in Africa, the Americas, Southeast Asia, and the Western Pacific region.

[0004] Chikungunya virus (CHIKV) is mainly transmitted among the population by Aedes mosquito bites. It was first discovered in Tanzania, Africa in 1952 and isolated from the blood of patients. Subsequently, the virus was successfully isolated from Aedes aegypti captured in the wild in 1956. CHIKV is mainly prevalent in Africa, South Asia, and Southeast Asia. As of now, CHIKV outbreaks have occurred in more than 100 countries and regions around the world.

[0005] Zika virus (ZIKV) has Aedes mosquitoes as the main transmission vector. It was first discovered in Uganda in 1947 and belongs to the genus Flavivirus. Since 2007, Zika virus has broken out and spread in regions such as the South Pacific, Africa, and Southeast Asia many times. As of now, more than 80 countries and regions around the world have reported Zika outbreaks. In 2016, it was reported that neonatal brain development disorders may be related to maternal infection with Zika virus during pregnancy, and subsequently Zika virus has attracted widespread international attention.

[0006] Yellow fever virus (YFV) can cause an acute infectious disease - yellow fever (YF). Aedes mosquitoes are the main transmission vector, and it is one of the three infectious diseases monitored by the International Health Regulations.

[0007] Zika virus, dengue virus, chikungunya virus and yellow fever virus not only share a common vector, but also have some overlap in their epidemic regions. They are characterized by fast transmission and easy prevalence. Once an outbreak occurs, it will seriously threaten people's health and social stability. In addition, in the early stage of patient infection and onset, the clinical symptoms caused by these four viruses are extremely similar, and even misleading, which brings difficulties to clinical differential diagnosis. Rapid diagnosis and appropriate treatment measures for patients in the early stage of the disease are important means to effectively prevent and control the occurrence and transmission of viral hemorrhagic fevers caused by the above-mentioned arboviruses.

[0008] At present, the diagnostic methods for the above viruses at home and abroad mainly rely on serological tests and tissue culture methods, which have disadvantages such as low sensitivity, immune cross-reaction and long cycle, so they are not suitable for early disease diagnosis. Molecular diagnostic techniques extract viral nucleic acids from respiratory samples, and then use primers and heat-stable polymerases to exponentially amplify the target nucleic acid fragments of the virus under appropriate conditions, thereby achieving viral nucleic acid detection. It has high sensitivity and specificity in virus detection and has early diagnostic value. Among them, real-time fluorescence quantitative PCR has great advantages in the early detection of viruses due to its rapid reaction, strong specificity, high sensitivity, etc. However, the molecular diagnostic industry has weaknesses such as slow speed and low automation. Without including the time for sample extraction and system configuration, a single PCR amplification often takes 1 to 2 hours. Existing technologies (such as CN113943836A, CN114507753A, CN102776297B, CN106086242A, etc.) adopt traditional primer design methods, which often take a lot of time for heating and cooling during actual amplification under conventional amplification procedures, so the actual detection time is often long, even exceeding 100 minutes. That is to say, the existing technologies using PCR to detect hemorrhagic fever pathogens often sacrifice detection speed to achieve the accuracy of detection results.

[0009] The speed of nucleic acid detection has to race with the virus. A faster detection speed can cut off the virus transmission chain earlier and minimize the scope of virus transmission. If sensitive and rapid diagnostic tests cannot be obtained (such as due to the long turnaround time of laboratory PCR results), medical practitioners usually make immediate treatment decisions based only on signs and clinical symptoms, which may misdiagnose patients and expand the spread of the virus. If patients infected with the above viruses still need to wait at least 1-2 hours for PCR results, it may seriously aggravate the patients' conditions and even pose a threat of death.

[0010] Currently, fast PCR is mainly achieved through the following aspects: 1) based on specific instruments, such as a PCR instrument with an improved temperature control module (for example, to increase the temperature change speed); 2) based on specific consumables, such as improved PCR tubes (for example, to reduce the PCR reaction volume to increase the temperature change speed); 3) based on optimized reagents, such as the screened fast Taq enzyme, and optimizing the reaction system into a system that can facilitate the realization of fast PCR.

[0011] In other words, the implementation of rapid PCR in the prior art requires specific enzymes, specific instruments and matching consumables. How to shorten the time of nucleic acid detection of hemorrhagic fever pathogens on a common instrument platform in the market (for example, based on the existing fluorescent quantitative PCR instrument) without changing the instrument hardware parameters, consumables and reaction reagents has become an urgent issue to be solved. Summary of the invention

[0012] In a first aspect, the present invention provides a rapid detection kit for multiple pathogens of hemorrhagic fever, characterized in that the kit comprises: an upstream primer for dengue virus, whose nucleotide sequence is shown in SEQ ID NO: 1; a downstream primer for dengue virus, whose nucleotide sequence is shown in SEQ ID NO: 3; a probe for detecting dengue virus, whose nucleotide sequence is shown in SEQ ID NO: 2;

[0013] An upstream primer for Chikungunya virus, whose nucleotide sequence is shown in SEQ ID NO:4; a downstream primer for Chikungunya virus, whose nucleotide sequence is shown in SEQ ID NO:6; a probe for detecting Chikungunya virus, whose nucleotide sequence is shown in SEQ ID NO:5;

[0014] An upstream primer for Zika virus, whose nucleotide sequence is shown in SEQ ID NO:7; a downstream primer for Zika virus, whose nucleotide sequence is shown in SEQ ID NO:9; a probe for detecting Zika virus, whose nucleotide sequence is shown in SEQ ID NO:8;

[0015] The nucleotide sequence of the upstream primer of yellow fever virus is shown in SEQ ID NO:10; the nucleotide sequence of the downstream primer of yellow fever virus is shown in SEQ ID NO:12; the nucleotide sequence of the probe for detecting yellow fever virus is shown in SEQ ID NO:11.

[0016] Under the design and exploration of the present invention, the primer sequences (and probe sequences) in the kit provided by the present invention not only ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection), but also have a higher annealing temperature. In conjunction with the corresponding PCR amplification program, it actually only takes about 24-35 minutes to achieve specific, accurate and sensitive rapid qualitative detection of the above-mentioned hemorrhagic fever pathogens.

[0017] Although the number of primers and probes in the kit provided by the present invention is relatively large, due to its relatively high annealing temperature, the formation of dimers between primers and (or) probes can be avoided to a certain extent during actual detection. Therefore, the kit provided by the present invention can simultaneously detect four arboviruses causing hemorrhagic fever (i.e., dengue virus, chikungunya virus, Zika virus, and yellow fever virus) in one tube and in one test, with low cost, high accuracy and sensitivity, and short time consumption. The actual detection time only takes about 24 - 35 min, and the test result can be determined by the Ct value. In addition, the whole detection process is carried out under the condition of single-tube closure, avoiding false positives and environmental pollution caused by cross-contamination between samples.

[0018] In some embodiments, the 5'-end of the probe sequence is labeled with a fluorescent group, and the fluorescent group includes HEX, FAM, ROX or CY5. In some embodiments, the fluorescent groups of each probe sequence should be different from each other and non-interfering, that is, the fluorescent group used for each probe sequence is different, and it will not affect the detection of each other, that is, detection can be carried out using different channels.

[0019] In some embodiments, the 3'-end of the probe sequence is labeled with a quenching group, and the quenching group includes BHQ1, BHQ3 or MGB.

[0020] In some embodiments, the 5'-end of the probe for detecting dengue virus is labeled with a FAM fluorescent group, and the 3'-end is labeled with an MGB quenching group; the 5'-end of the probe for detecting chikungunya virus is labeled with a HEX fluorescent group, and the 3'-end is labeled with a BHQ1 quenching group; the 5'-end of the probe for detecting Zika virus is labeled with a CY5 fluorescent group, and the 3'-end is labeled with a BHQ3 quenching group; the 5'-end of the probe for detecting yellow fever virus is labeled with a ROX fluorescent group, and the 3'-end is labeled with an MGB quenching group.

[0021] In some embodiments, the kit further includes: DNA polymerase, reverse transcriptase, and UDG enzyme.

[0022] In some embodiments, the kit further includes: a positive control product, and the positive control product includes pseudoviruses containing target fragments of dengue virus, chikungunya virus, Zika virus, and yellow fever virus.

[0023] In some embodiments, the kit further includes: a negative control product, and the negative control product includes physiological saline.

[0024] In some embodiments, the kit further includes: PCR buffer.

[0025] In some embodiments, the kit further includes: dNTP.

[0026] In some embodiments, the kit is used for fluorescence quantitative PCR, which includes a first amplification stage and a second amplification stage. The first amplification conditions of the first amplification stage include a denaturation temperature of 90 - 99°C, an annealing temperature of 55 - 65°C, and a cycle number of 3 - 15 in each cycle; the second amplification conditions of the second amplification stage include a denaturation temperature of at least 85°C, an annealing temperature of 66 - 82°C, and a cycle number of 15 - 50 in each cycle.

[0027] In some embodiments, the specific conditions of the fluorescence quantitative PCR include: the first amplification conditions include 92 - 95°C for 1 - 10 s, 56 - 62°C for 10 - 15 s, and 3 - 10 cycles; the second amplification conditions include 92 - 95°C for 1 - 10 s, 68 - 72°C for 10 - 15 s, and 35 - 45 cycles.

[0028] When the nucleic acid concentration in the sample is relatively high and / or there are more stringent requirements for the detection speed, the specific conditions of the fluorescence quantitative PCR can be set as follows: the first amplification conditions include 92°C for 1 s, 62°C for 10 s, and 3 cycles; the second amplification conditions include 92°C for 1 s, 72°C for 10 s, and 35 cycles. After testing, under the above - mentioned condition settings, the kit provided by the present invention actually only needs 24 min (including the reverse transcription and DNA polymerase enzyme activation steps, with reverse transcription set at 50°C for 2 min and enzyme activation set at 98°C for 30 s, using the Bio - Rad 96C PCR instrument) to achieve rapid and accurate detection of the above - mentioned hemorrhagic fever pathogen.

[0029] In some embodiments, the specific conditions of the fluorescence quantitative PCR can be set as follows: the first amplification conditions include 92°C for 1 s, 60°C for 10 s, and 5 cycles; the second amplification conditions include 92°C for 1 s, 70°C for 10 s, and 40 cycles. After testing, under the above - mentioned condition settings, the kit provided by the present invention actually only needs 29 min (including the reverse transcription and DNA polymerase enzyme activation steps, with reverse transcription set at 50°C for 2 min and enzyme activation set at 98°C for 30 s, using the Bio - Rad 96C PCR instrument) to achieve rapid and accurate detection of the above - mentioned hemorrhagic fever pathogen.

[0030] In some embodiments, the actual detection time of the kit is 24 - 35 min. Those skilled in the art should understand that the specific conditions of the fluorescence quantitative PCR can be adjusted according to the actual detection requirements and actual detection scenarios (for example, by extending the denaturation / annealing time, etc.).

[0031] Second, the present invention also provides a method for detecting hemorrhagic fever pathogen for non - diagnostic purposes, which is characterized by including:

[0032] 1) Extract viral nucleic acid from the sample;

[0033] 2) Perform fluorescence quantitative PCR on the viral nucleic acid using the above kit;

[0034] 3) Obtain and analyze the results;

[0035] Wherein the fluorescence quantitative PCR includes a first amplification stage and a second amplification stage. The first amplification conditions of the first amplification stage include a denaturation temperature of 90 - 99 °C, an annealing temperature of 55 - 65 °C, and a cycle number of 3 - 15 in each cycle; the second amplification conditions of the second amplification stage include a denaturation temperature of at least 85 °C, an annealing temperature of 66 - 82 °C, and a cycle number of 15 - 50 in each cycle.

[0036] In some embodiments, the specific conditions of the fluorescence quantitative PCR include: the first amplification conditions include 92 - 95 °C for 1 - 10 s, 56 - 62 °C for 10 - 15 s, and 3 - 10 cycles; the second amplification conditions include 92 - 95 °C for 1 - 10 s, 68 - 72 °C for 10 - 15 s, and 35 - 45 cycles.

[0037] In some embodiments, the fluorescence quantitative PCR further includes steps of reverse transcription and DNA polymerase enzyme activation. The specific conditions of the reverse transcription and DNA polymerase enzyme activation include 50 - 55 °C for 2 - 5 min, 95 - 98 °C for 30 - 120 s.

[0038] In some embodiments, the sample includes one or more of serum, urine, and saliva.

[0039] In some embodiments, the virus concentration includes 250–10 7 copies / mL.

[0040] In some embodiments, when a typical S-shaped amplification curve appears in the detection channel and the Ct value of the amplification curve ≤ 39, it indicates that the sample contains the corresponding viral nucleic acid, and the sample is evaluated as a positive result; when there is no amplification curve in the detection channel or the Ct value > 39, it indicates that the corresponding viral nucleic acid is not detected in the sample, and the sample is evaluated as a negative result.

[0041] Beneficial effects

[0042] The kit of the present invention is based on the TaqMan probe-based real-time fluorescence quantitative PCR technology. A conserved sequence of hemorrhagic fever virus is selected as the target detection gene, and specific primers and probes are designed. Compared with the existing kits for detecting hemorrhagic fever pathogens, the kit provided by the present invention does not require modification of the probes and primers, nor the addition of specific enzymes and reagents to the reaction system. It can rapidly and accurately detect and distinguish four arboviruses causing hemorrhagic fever (i.e., dengue virus, chikungunya virus, Zika virus, and yellow fever virus) on commonly used fluorescence quantitative PCR instruments on the market. The actual detection time is much lower than that of other kits based on qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction) technology.

[0043] Timely diagnosis is the key to preventing the spread and outbreak of hemorrhagic fever pathogens. The kit provided by the present invention can simultaneously, rapidly, and accurately detect four arboviruses causing hemorrhagic fever (i.e., dengue virus, chikungunya virus, Zika virus, and yellow fever virus). The actual detection time only takes 24 - 35 minutes, which is particularly suitable for scenarios such as POC testing (Point-of-Care Testing), outpatient clinics, emergency departments, and customs where rapid test results are required. Compared with the existing kits, the kit provided by the present invention can help determine whether a suspected case or a suspected vector is infected with the above-mentioned arboviruses in a shorter time, which not only helps reduce the outbreak frequency and scale of arboviruses, but also helps prevent and control the occurrence and spread of hemorrhagic fever at the source. It is applicable to the early diagnosis, effective monitoring, and control of viral hemorrhagic fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally denoted by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0045] Figure 1 It is a schematic diagram of the first amplification stage and the second amplification stage of the present invention;

[0046] Figure 2 It is a schematic diagram of the structure of the primer of the present invention;

[0047] Figure 3 It is the amplification curve of the DENV conventional procedure;

[0048] Figure 4 Amplification curve for the DENV rapid procedure;

[0049] Figure 5 Amplification curve for the CHIKV conventional procedure;

[0050] Figure 6 Amplification curve for the CHIKV rapid procedure;

[0051] Figure 7 Amplification curve for the ZIKV conventional procedure;

[0052] Figure 8 Amplification curve for the ZIKV rapid procedure;

[0053] Figure 9 Amplification curve for the YFV rapid procedure;

[0054] Figure 10 Amplification curve for the dengue virus sensitivity experiment;

[0055] Figure 11 Amplification curve for the chikungunya virus sensitivity experiment;

[0056] Figure 12 Amplification curve for the Zika virus sensitivity experiment;

[0057] Figure 13 Amplification curve for the yellow fever virus sensitivity experiment;

[0058] Figure 14 Amplification curve for the dengue virus repeatability experiment;

[0059] Figure 15 Amplification curve for the chikungunya virus repeatability experiment;

[0060] Figure 16 Amplification curve for the Zika virus repeatability experiment;

[0061] Figure 17 Amplification curve for the yellow fever virus repeatability experiment. Detailed implementation manners

[0062] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0063] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0064] As used herein, "a plurality of" means two or more, i.e., it includes two, three, four, five, etc.

[0065] It should be noted that, as used herein, the terms "comprising", "including" or any other variation thereof are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or apparatus comprising that element.

[0066] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0067] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range 1 - 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0068] Example 1

[0069] 1. The kit of the present invention is composed as follows:

[0070] 2. Reagent detection process

[0071] 1) Nucleic acid extraction: Nucleic acid is extracted from the sample to be tested. Nucleic acid extraction can be performed using a conventional commercial kit.

[0072] 2) Preparation of PCR amplification reagents, and the amplification system is prepared as follows: Reaction solution A Reaction solution B 16 μL 2 μL

[0073] The present invention has no restrictive requirements for the amplification enzyme mix and buffer, and Taq enzyme can be selected according to the actual situation (such as Taq enzyme from companies like Finney Biotech Co., Ltd. and Novoprotein).

[0074] 3. PCR Amplification

[0075] The present invention has no restrictive requirements for real-time fluorescence quantitative PCR instruments, and a suitable PCR instrument can be selected according to the actual situation, such as ABI 7500, ABI Q5, Hongshi SLAN, Anyu Technology AGS, Tianlong Technology Gentier 96E, Bori Technology 96C, etc.

[0076] The PCR amplification experiment of the present invention is divided into two rounds of amplification (after the reverse transcription and pre-denaturation steps are completed), see Figure 1 . See the primer design structure diagram in Figure 2 .

[0077] 1) First round of amplification (first amplification stage): Amplification of the sequence binding to the template (forward primer / reverse primer), the annealing temperature is generally about 60°C, and generally 3 - 15 cycles are used in this stage, and fluorescence is not collected.

[0078] 2) Second round of amplification (second amplification stage): Amplification of the full-length primer (adapter sequence + forward primer / reverse primer), the annealing temperature is generally about 70°C, and generally 15 - 50 cycles are used in this stage, and fluorescence is collected.

[0079] 4. Result Analysis

[0080] After the reaction ends, the results are automatically saved. Adjust the Start value, End value, and Threshold value of Baseline according to the analyzed image (users can adjust according to the actual situation, the Start value can be set at 3 - 15, the End value can be set at 5 - 20, and set the Value value of Threshold in the Log graph window so that the threshold line is located in the exponential phase of the amplification curve, and the amplification curve of the negative control product is flat or below the threshold line), and click analysis to automatically obtain the analysis results.

[0081] Example 2

[0082] 1. Primer Information

[0083] According to the primer and probe sequences (Table 1), PCR amplification conditions (Table 3), and the corresponding experimental parameters mentioned in "Establishment and Preliminary Evaluation of Zika Virus - related Nucleic Acid Detection Methods" (Master's degree thesis of the Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Gong Xuerui) (abbreviated as Document 1 in this example), the actual amplification time on the Bori Technology 96C PCR instrument is 108 minutes.

[0084] Table 1

[0085] The present invention designs rapid primers for four pathogens, namely dengue virus, chikungunya virus, Zika virus and yellow fever virus (as shown in Table 2), wherein the primers are partially paired with the template.

[0086] Table 2 Note: Italics indicate the adapter sequence, and the underlined part indicates the sequence in the adapter sequence that does not match the template.

[0087] The target sequences of the above rapid primers are as follows:

[0088] 2. Nucleic acid extraction

[0089] Nucleic acid extraction is carried out on the dengue virus pseudovirus, chikungunya virus pseudovirus, Zika virus pseudovirus, and yellow fever virus pseudovirus serially diluted with healthy human serum, with two replicates for each dilution.

[0090] 3. PCR amplification system

[0091] The following system is prepared for the extracted RNA:

[0092] 1) The PCR amplification system of the conventional primers (the system in Document 1) is shown in Table 3 below:

[0093] Table 3 Component Sample loading volume (μL) Final concentration / reaction Enzyme mixture 2 / 2X Buffer 12.5 1× DENV-F (20 μM) 0.25 0.2 μM DENV-R (20 μM) 0.25 0.2 μM DENV-P (20 μM) 0.125 0.1 μM CHIKV-F (20 μM) 0.25 0.2 μM CHIKV-R (20 μM) 0.25 0.2 μM CHIKV-P (20 μM) 0.125 0.1 μM ZIKV-F (20 μM) 0.25 0.2 μM ZIKV-R (20 μM) 0.25 0.2 μM ZIKV-P (20 μM) 0.125 0.1 μM <![CDATA[DEPC H 2 O]]> 2.375 / dNTP (10 mM) 0.25 0.1 mM <![CDATA[MgCl 2 (25 mM)]]> 1 1 mM Template 5 μL / Total volume 25 μL /

[0094] 2) The rapid program PCR amplification system of this example is shown in Table 4 below:

[0095] Table 4

[0096] 4. PCR amplification program

[0097] 1) The conventional program (Document 1) is shown in Table 5:

[0098] Table 5

[0099] 2) The rapid primer program of this example is shown in Table 6:

[0100] Table 6

[0101] After actual testing, the primers and probes of Document 1, the amplification conditions, the mentioned experimental parameters, and the kit and rapid amplification program of the present invention are compared in terms of the actual amplification time on the Bori Technology 96C and ABI Q5 as follows. The results show that the kit of the present invention can achieve rapid detection on different fluorescence quantitative PCR instruments, and the actual detection time is much shorter than that of conventional primers.

[0102] 5. Analysis of experimental results

[0103] The following experiments were all completed on the ABI Q5 instrument. The results were automatically saved after the reaction. The Start value, End value, and Threshold value of Baseline were adjusted according to the analyzed image, and the analysis results were automatically obtained by clicking analysis. The results are as shown in Table 7 and Figures 3 - 9 As shown, the results indicate that the rapid primers of the present invention can achieve an amplification effect similar to that of existing conventional primers and conventional procedures, but the actual required PCR amplification time is much shorter than that of conventional primers and conventional procedures.

[0104] Figure 3 Among them, there are 10 amplification curves from left to right. The two closest ones are the amplification curves of 2 replicates of the same concentration DENV sample in the conventional procedure. The concentrations corresponding to the amplification curves are 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL. Figure 4 Among them, there are 10 amplification curves from left to right. The two closest ones are the amplification curves of 2 replicates of the same concentration DENV sample in the rapid procedure of the present invention. The concentrations corresponding to the amplification curves are 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL. Figure 5 Among them, there are 10 amplification curves from left to right. The two closest ones are the amplification curves of 2 replicates of the same concentration CHIKV sample in the conventional procedure. The concentrations corresponding to the amplification curves are 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4copies / mL, 1×10 3 copies / mL. Figure 6 In it, there are 10 amplification curves from left to right. The two closest ones are the amplification curves of 2 replicates of the same concentration CHIKV sample in the rapid procedure of the present invention. The concentrations corresponding to the amplification curves are 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL. Figure 7 In it, there are 10 amplification curves from left to right. The two closest ones are the amplification curves of 2 replicates of the same concentration ZIKV sample in the conventional procedure. The concentrations corresponding to the amplification curves are 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL. Figure 8 In it, there are 10 amplification curves from left to right. The two closest ones are the amplification curves of 2 replicates of the same concentration ZIKV sample in the rapid procedure of the present invention. The concentrations corresponding to the amplification curves are 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL. Figure 9 In it, there are 10 amplification curves from left to right. The two closest ones are the amplification curves of 2 replicates of the same concentration YFV sample in the rapid procedure of the present invention. The concentrations corresponding to the amplification curves are 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL.

[0105] Table 7 (Ct value) Note: * Fluorescence is not collected in the first 5 cycles of the rapid procedure. The Ct values in the table are the Ct values exported by the instrument + 5.

[0106] Example 3

[0107] Sensitivity detection

[0108] Dilute dengue virus pseudovirus, chikungunya virus pseudovirus, Zika virus pseudovirus, and yellow fever virus pseudovirus to 500 copies / mL, and perform 20 repeated detections using the kit of the present invention respectively. The results are as shown in Table 8 below and Figures 10 - 13 As shown, the kits of the present invention can all detect them, and it can be determined that the sensitivity of the kits of the present invention is not lower than 500 copies / mL. Figure 10 Among them, there are 20 amplification curves from left to right, and the concentration of dengue virus pseudovirus corresponding to the amplification curve is 500 copies / mL. Figure 11 Among them, there are 20 amplification curves from left to right, and the concentration of chikungunya virus pseudovirus corresponding to the amplification curve is 500 copies / mL. Figure 12 Among them, there are 20 amplification curves from left to right, and the concentration of Zika virus pseudovirus corresponding to the amplification curve is 500 copies / mL. Figure 13 Among them, there are 20 amplification curves from left to right, and the concentration of yellow fever virus pseudovirus corresponding to the amplification curve is 500 copies / mL.

[0109] Table 8: Statistical results of Ct values for 20 repeated detections in the sensitivity experiment Number of replicates DENV CHIKV ZIKV YFV 1 35.78 37.72 38.94 37.27 2 36.38 37.26 38.36 37.12 3 35.09 36.75 37.95 37.26 4 35.44 37.48 38.04 36.18 5 34.91 36.00 38.00 36.85 6 35.79 36.18 37.79 36.12 7 36.40 36.81 37.82 37.35 8 35.85 36.69 38.18 36.25 9 36.20 37.56 38.88 37.45 10 36.33 36.08 37.32 36.44 11 35.81 35.81 38.52 36.62 12 35.70 38.45 37.55 37.70 13 35.53 35.62 38.28 36.97 14 36.18 37.01 37.74 36.29 15 35.68 35.75 38.81 36.89 16 35.86 35.04 38.20 37.40 17 35.64 37.53 38.25 37.42 18 35.25 37.85 37.84 37.23 19 35.80 37.05 37.68 36.74 20 35.51 36.11 38.50 37.16

[0110] Example 4

[0111] Specificity detection

[0112] The viruses for the cross-reaction experiment are parainfluenza virus, influenza A virus, influenza B virus, measles virus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and severe fever with thrombocytopenia syndrome virus. Nucleic acid extraction and amplification are performed on the above virus cultures / samples. The results are shown in Table 9. Using the kit provided by the present invention to detect the above pathogens, there is no cross-reaction.

[0113] Table 9

[0114] Example 5:

[0115] Repeatability detection

[0116] Samples with high, medium, and low concentrations of dengue virus, chikungunya virus, Zika virus, and yellow fever virus were respectively verified. The low concentration was near the lowest detection limit, and each concentration was repeatedly detected 20 times. The calculated coefficient of variation (CV) was < 5% (even less than 1.3%). The results are shown in Table 10 and Figures 14 - 17 as follows, indicating that the kit provided by the present invention has good repeatability. Figure 14 There are a total of 60 amplification curves from left to right. The closest 20 are the amplification curves of the same concentration DENV samples. The corresponding high, medium, and low concentrations of the amplification curves are 5×10 6 copies / mL, 5×10 4 copies / mL, 5×10 2 copies / mL. Figure 15 There are a total of 60 amplification curves from left to right. The closest 20 are the amplification curves of the same concentration CHIKV samples. The corresponding high, medium, and low concentrations of the amplification curves are 1×10 7 copies / mL, 1×10 5 copies / mL, 1×10 3 copies / mL. Figure 16 There are a total of 60 amplification curves from left to right. The closest 20 are the amplification curves of the same concentration ZIKV samples. The corresponding high, medium, and low concentrations of the amplification curves are 1×10 7 copies / mL, 1×10 5 copies / mL, 1×10 3 copies / mL. Figure 17 There are a total of 60 amplification curves from left to right. The closest 20 are the amplification curves of the same concentration YFV samples. The corresponding high, medium, and low concentrations of the amplification curves are 5×10 6 copies / mL, 5×10 4 copies / mL, 5×10 2 copies / mL.

[0117] Table 10

[0118] Example 6:

[0119] Clinical sample detection

[0120] The nucleic acids of 10 clinical samples were detected using the kit of the present invention, and the results are shown in Table 11 below. When a typical S-shaped amplification curve appears in the detection channel and the Ct value of the amplification curve ≤ 39, it indicates that the sample contains the corresponding viral nucleic acid, and the sample is evaluated as a positive result; when there is no amplification curve in the detection channel or the Ct value > 39, it indicates that the corresponding viral nucleic acid is not detected in the sample, and the sample is evaluated as a negative result. The results show that the kit of the present invention can be applied to the detection of clinical samples, and the detection results are all consistent with the expectations.

[0121] Table 11

[0122] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims. All of these fall within the protection scope of the present invention.

Claims

1. Rapid detection kit for multiple pathogens of hemorrhagic fever, characterized in that, the kit includes: upstream primer of dengue virus, whose nucleotide sequence is shown as SEQ ID NO:1; downstream primer of dengue virus, whose nucleotide sequence is shown as SEQ ID NO:3; probe for detecting dengue virus, whose nucleotide sequence is shown as SEQ ID NO:2; upstream primer of chikungunya virus, whose nucleotide sequence is shown as SEQ ID NO:4; downstream primer of chikungunya virus, whose nucleotide sequence is shown as SEQ ID NO:6; probe for detecting chikungunya virus, whose nucleotide sequence is shown as SEQ ID NO:5; upstream primer of Zika virus, whose nucleotide sequence is shown as SEQ ID NO:7; downstream primer of Zika virus, whose nucleotide sequence is shown as SEQ ID NO:9; probe for detecting Zika virus, whose nucleotide sequence is shown as SEQ ID NO:8; upstream primer of yellow fever virus, whose nucleotide sequence is shown as SEQ ID NO:10; downstream primer of yellow fever virus, whose nucleotide sequence is shown as SEQ ID NO:12; probe for detecting yellow fever virus, whose nucleotide sequence is shown as SEQ ID NO:

11.

2. The kit according to claim 1, characterized in that, the kit further includes: DNA polymerase, reverse transcriptase, UDG enzyme.

3. The kit according to claim 1, characterized in that, the kit further includes: positive control product, and the positive control product includes pseudoviruses containing target fragments of dengue virus, chikungunya virus, Zika virus and yellow fever virus.

4. The kit according to claim 1, characterized in that, the 5' end of the probe sequence is labeled with a fluorescent group, and the fluorescent group includes HEX, FAM, ROX or CY5.

5. The kit according to claim 1, characterized in that, the 3' end of the probe sequence is labeled with a quenching group, and the quenching group includes BHQ1, BHQ3 or MGB.

6. The kit according to claim 1, characterized in that, the 5' end of the probe for detecting dengue virus is labeled with FAM fluorescent group, and the 3' end is labeled with MGB quenching group; the 5' end of the probe for detecting chikungunya virus is labeled with HEX fluorescent group, and the 3' end is labeled with BHQ1 quenching group; the 5' end of the probe for detecting Zika virus is labeled with CY5 fluorescent group, and the 3' end is labeled with BHQ3 quenching group; the 5' end of the probe for detecting yellow fever virus is labeled with ROX fluorescent group, and the 3' end is labeled with MGB quenching group.

7. The kit according to claim 1, characterized in that, The kit is used for fluorescence quantitative PCR, and the fluorescence quantitative PCR includes a first amplification stage and a second amplification stage. The first amplification conditions of the first amplification stage include that the denaturation temperature in each cycle is 90-99°C, the annealing temperature is 55-65°C, and the number of cycles is 3-15; the second amplification conditions of the second amplification stage include that the denaturation temperature in each cycle is at least 85°C, the annealing temperature is 66-82°C, and the number of cycles is 15-50.

8. A method for detecting hemorrhagic fever pathogen for non-diagnostic purposes, characterized in that, comprising: 1) Extracting viral nucleic acid from a sample; 2) Performing fluorescence quantitative PCR on the viral nucleic acid using the kit according to any one of claims 1-7; 3) Obtaining and analyzing the results; wherein the fluorescence quantitative PCR includes a first amplification stage and a second amplification stage. The first amplification conditions of the first amplification stage include that the denaturation temperature in each cycle is 90-99°C, the annealing temperature is 55-65°C, and the number of cycles is 3-15; the second amplification conditions of the second amplification stage include that the denaturation temperature in each cycle is at least 85°C, the annealing temperature is 66-82°C, and the number of cycles is 15-50.

9. The method according to claim 8, characterized in that, the specific conditions of the fluorescence quantitative PCR include: the first amplification conditions include 92-95°C for 1-10 s, 56-62°C for 10-15 s, and 3-10 cycles; the second amplification conditions include 92-95°C for 1-10 s, 68-72°C for 10-15 s, and 35-45 cycles.

10. The method according to claim 8, characterized in that, the fluorescence quantitative PCR further includes steps of reverse transcription and DNA polymerase enzyme activation, and the specific conditions of the reverse transcription and DNA polymerase enzyme activation include 50-55°C for 2-5 min, 95-98°C for 30-120 s.

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