Compositions, kits and uses for detecting hemorrhagic fever-associated pathogens

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to the detection of hemorrhagic fever infection related pathogens, and more particularly to the detection of Marburg virus, Rift Valley fever virus, Chikungunya virus and Alkuma hemorrhagic fever virus. The combined composition provided by the present application mainly utilizes a multiplex fluorescence PCR analysis method, and different pathogens are detected by detecting target points on different pathogens, so that the detection and differentiation of Marburg virus, Rift Valley fever virus, Chikungunya virus and Alkuma hemorrhagic fever virus are simultaneously realized in a single tube reaction system. The composition of the present application has higher sensitivity, reaches 400 copies / mL, has good specificity, and is more accurate in detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically, it relates to the detection of hemorrhagic fever-related pathogens, and more specifically, it relates to Marburg virus, Rift Valley fever virus, Chikungunya virus, and Alkuma hemorrhagic fever virus. Background Technology

[0002] Marburg hemorrhagic fever (MHF) is a severe acute viral infectious disease caused by Marburg virus (MBGV), with a mortality rate as high as 88%. MHF often presents suddenly, with early symptoms including fever, chills, headache, painful swallowing, muscle pain, vomiting, and diarrhea, which are not particularly specific. Death usually occurs within a week of onset. Marburg virus is transmitted to humans through fruit bats and spreads from person to person through bodily fluids, including blood, excrement, saliva, and vomit, leading to highly contagious hemorrhagic fever. Marburg virus most easily infects children; in Africa, 75% of cases occur in children under 5 years old. Adult infections are mostly close relatives and healthcare workers who have had close contact with infected children. Currently, there is no specific treatment for this highly contagious and fatal disease; management relies mainly on early detection, early isolation, symptomatic treatment, and aggressive supportive care.

[0003] Rift Valley fever is an acute viral infection caused by the Rift Valley fever virus (RVFV), transmitted through mosquitoes or contact. Infection can cause hemorrhagic fever, hepatitis, and encephalitis, and in severe cases, it can lead to death. Clinical features include sudden onset of fever (often biphasic), headache, fatigue, and joint and muscle pain. Severe cases may lead to hemorrhage, shock, encephalitis, hepatitis, or even death.

[0004] Chikungunya fever is caused by the chikungunya virus (CHIKV) and transmitted by Aedes mosquitoes. It is an acute infectious disease characterized by muscle pain, headache, nausea, fatigue, and rash, with fever, rash, and joint pain / arthritis as the main symptoms. In rare cases, meningoencephalitis, liver damage, myocarditis, and skin and mucous membrane bleeding may occur. Chikungunya fever is mainly distributed in Africa, South Asia, and Southeast Asia. From 2005 to 2007, the disease was widespread in the Indian Ocean islands, India, and Southeast Asia, causing millions of cases. Because infection usually presents with only mild symptoms, it is difficult to identify based on clinical manifestations alone, and it is often misdiagnosed in dengue-endemic areas.

[0005] Alkhurma hemorrhagic fever (AHFV) is caused by the Alkhurma hemorrhagic fever virus (AHFV) and is transmitted through tick bites or contact with the blood of infected livestock. Initial symptoms include fever, headache, diarrhea, vomiting, muscle and joint pain, loss of appetite, and chills. Some patients develop neurological hemorrhage and multiple organ failure before death. Currently, there is no specific treatment or vaccine for AHFV, and the virus has a potential impact on regional public health.

[0006] Marburg virus, Rift Valley fever virus, Chikungunya virus, and Alkuma hemorrhagic fever virus can all cause viral hemorrhagic fevers, and their clinical presentations are quite similar. The main symptoms include fever, fatigue, headache, and muscle aches. These symptoms may progress to vascular leakage, bleeding, and multiple organ failure. For infected patients, early diagnosis and treatment are crucial, along with timely control of the source of infection and interruption of transmission routes. Establishing a rapid diagnostic method for Marburg virus, Rift Valley fever virus, Chikungunya virus, and Alkuma hemorrhagic fever virus is of great significance for the differential diagnosis of hemorrhagic fever-related viruses and the prevention and control of outbreaks.

[0007] Therefore, there is a need in this field for a product that can easily and quickly detect the above-mentioned pathogens with high sensitivity and specificity, so as to provide clinicians with sufficient and rapid diagnostic evidence and to rule out infection by different pathogens, shorten the time for clinicians to diagnose patients' conditions, and speed up the implementation of treatment measures for patients. Summary of the Invention

[0008] In view of this, in a first aspect, the present invention provides a composition for detecting hemorrhagic fever infection pathogens, comprising:

[0009] The upstream primers, downstream primers, and probes for detecting Marburg virus are shown in SEQ ID NO:1-3;

[0010] For example, the upstream primers, downstream primers and probes for detecting Rift Valley fever virus shown in SEQ ID NO:4-6;

[0011] For example, the upstream primers, downstream primers, and probes for detecting Chikungunya virus shown in SEQ ID NO:7–9; and

[0012] The upstream primers, downstream primers, and probes for detecting Alkumar hemorrhagic fever virus are shown in SEQ ID NO:10-12.

[0013] The combined detection composition provided by this invention primarily utilizes multiplex fluorescent PCR analysis to detect different pathogens by detecting target sites on them. This allows for the simultaneous detection and differentiation of Marburg virus, Rift Valley fever virus, Chikungunya virus, and Alkuma hemorrhagic fever virus in a single-tube reaction system, providing targeted strategies for subsequent treatment. The composition of this invention offers higher detection sensitivity (up to 400 copies / mL), better specificity, and more accurate detection, providing clinicians with sufficient and rapid diagnostic evidence to rule out different pathogen infections, shortening the time required for diagnosis and accelerating treatment.

[0014] Furthermore, the fluorescent groups of the probes in the composition of the present invention are different from each other and do not interfere with each other.

[0015] In this article, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorophore and will not affect the detection of each other, i.e., different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values ​​and can be selected in different channels, thus avoiding mutual interference.

[0016] In some specific implementations, the fluorescent reporter group for the Marburg virus probe is FAM; the fluorescent reporter group for the Rift Valley fever virus probe is ROX; the fluorescent reporter group for the Chikungunya virus probe is CY5; and the fluorescent reporter group for the Alkuma hemorrhagic fever virus probe is HEX (or VIC).

[0017] Furthermore, in some embodiments, the composition of the present invention may simultaneously include one or more pairs of the primer and probe pairs described above. In the present invention, a "pair" refers to a mutually matched upstream and downstream primer and probe for detecting a target.

[0018] The compositions of this invention can be arbitrarily combined to detect any combination of four corresponding targets. Those skilled in the art can combine them as needed, determining which targets to detect by combining the primer and probe pairs corresponding to those targets. All such combinations are included in this invention.

[0019] For example, it can include any 3 pairs of the above 4 pairs of primers and probes, it can include any 2 pairs of the above 4 pairs of primers and probes, or it can include any 1 pair of the above 4 pairs of primers and probes.

[0020] In some specific embodiments, the composition further includes upstream primers, downstream primers, and probes for detecting the internal standard.

[0021] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0022] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0023] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.

[0024] In one specific implementation, the 3' end of the probe is BHQ1.

[0025] In one specific embodiment, each component of the composition of the present invention is contained in a separate package.

[0026] In one specific embodiment, the components of the composition of the present invention are contained in the same package.

[0027] Furthermore, the components of the composition of the present invention exist in a mixed form.

[0028] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for detecting pathogens of hemorrhagic fever infection, wherein the pathogen is Marburg virus, Rift Valley fever virus, Chikungunya virus and / or Alkuma hemorrhagic fever virus.

[0029] Thirdly, the present invention provides a kit for detecting hemorrhagic fever pathogens, the kit comprising the composition of the present invention as described above.

[0030] Furthermore, the kit also includes negative and positive controls.

[0031] In one specific implementation scheme, the negative control is at least one of DEPC H2O and physiological saline. The positive control is at least one of a pseudovirus or fragment gene of Marburg virus, Rift Valley fever virus, Chikungunya virus, or Alkuma hemorrhagic fever virus.

[0032] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0033] Furthermore, the kit also includes at least one of the following: nucleic acid release reagent, nucleic acid extraction reagent, reverse transcriptase, and DNA polymerase.

[0034] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, reverse transcriptase, DNA polymerase, PCR buffer, and Mg2+. 2+ At least one of them.

[0035] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction; for example, the DNA polymerase may be Taq polymerase. The concentration of the reverse transcriptase is 0.1 U / reaction to 3 U / reaction.

[0036] In one specific embodiment, the kit of the present invention includes Taq enzyme, RT enzyme, and Mg 2+ dNTPs, primers, probes, and PCR buffer.

[0037] The PCR buffer components include Tris-HCl, KCl, and Triton X-100, with a total volume of 20 μl to 200 μl in a single PCR reaction tube.

[0038] Fourthly, a method for detecting hemorrhagic fever pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

[0039] 1) Extract nucleic acid from the sample to be tested;

[0040] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0041] 3) Obtain and analyze the results.

[0042] In this invention, the sample used for detection can be plasma or serum, but is not limited thereto.

[0043] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0044] Reverse transcription reaction: temperature 50–60℃, time 1–6 min, 1 cycle; cDNA pre-denaturation: temperature 95℃, time 1–60 seconds, 1 cycle; denaturation: temperature 95℃, time 5–20 seconds; annealing: temperature 55℃–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0045] In one specific embodiment, the use of a composition for preparing a reagent for detecting hemorrhagic fever infection pathogens is provided, the detection comprising the following steps:

[0046] 1) Extract nucleic acid from the sample to be tested;

[0047] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition or kit of the present invention as described above;

[0048] 3) Obtain and analyze the results.

[0049] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0050] Reverse transcription reaction: temperature 50–60℃, time 1–6 min, 1 cycle; cDNA pre-denaturation: temperature 95℃, time 1–60 seconds, 1 cycle; denaturation: temperature 95℃, time 5–20 seconds; annealing: temperature 55℃–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0051] In this article, the term "non-diagnostic purpose" refers to something not intended to obtain information about whether an individual is infected with the aforementioned pathogens and has contracted hemorrhagic fever. For example, the presence of the aforementioned pathogens may be required in test cultures (e.g., plasma or serum). Attached Figure Description

[0052] Figure 1 The combined detection results of the composition of the present invention (Marburg virus, Rift Valley fever virus, Chikungunya virus, Alkuma hemorrhagic fever virus);

[0053] Figures 2-5 The sensitivity results of the compositions of the present invention are shown in the graphs (for Marburg virus, Alkuma hemorrhagic fever virus, Rift Valley fever virus, and Chikungunya virus, respectively).

[0054] Figure 6 This is a graph showing the specificity of the composition of the present invention;

[0055] Figure 7 This is a graph showing the precision results of the composition of the present invention;

[0056] Figures 8-11 The images show the single-detection results of the comparative composition of the present invention (representing Marburg virus, Alkuma hemorrhagic fever virus, Rift Valley fever virus, and Chikungunya virus, respectively).

[0057] Figure 12 This is a graph showing the results of a quadruple assay of the comparative composition of the present invention. Detailed Implementation

[0058] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0059] Example 1: Primers and probes used in this invention

[0060] The primers and probes used in this invention are shown in Table 1 below:

[0061] Table 1

[0062]

[0063] The fluorescent reporter group for the Marburg virus probe is FAM; the fluorescent reporter group for the Rift Valley fever virus probe is ROX; the fluorescent reporter group for the Chikungunya virus probe is CY5; and the fluorescent reporter group for the Alkuma hemorrhagic fever virus probe is HEX.

[0064] Example 2: Method for detecting pathogens

[0065] The sample to be tested in this invention is prepared with the following reagents:

[0066] Based on the number of samples to be tested, positive controls, and negative controls, take the corresponding amounts of PCR reaction solution and enzyme mixture according to the ratio (37.5 μL / person PCR reaction solution + 2.5 μL / person enzyme mixture), mix thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds, and set aside for later use.

[0067] Sample processing and loading

[0068] Nucleic acid extraction was performed using the nucleic acid extraction or purification reagent (S10015) from Sansure Biotech Inc., following its instructions.

[0069] Take 10 μL each of the above-processed sample, negative control, and positive control and add them to the corresponding 0.2 mL PCR reaction tubes. Add 40 μL of PCR mixture to each tube and cap the tubes.

[0070] The real-time fluorescence PCR reaction system was prepared according to Table 2 below:

[0071] Table 2

[0072] PCR buffer 35.4μL RT enzyme (5 U / μL) 0.5μL Taq enzyme (5 U / μL) 2μL <![CDATA[Mg 2+ (1M)]]> 0.1μL dNTP (0.7mM) 1.2μL Primer (150 nM) 0.6μL Probe (150 nM) 0.2μL Sample DNA 10μL

[0073] The PCR amplification program should be set as shown in Table 3 below:

[0074] Table 3

[0075]

[0076] Results analysis:

[0077] 1) The target detection signals are FAM, HEX (or VIC), ROX, and CY5;

[0078] 2) Baseline settings: The baseline is generally set to 3-15 cycles, which can be adjusted according to the actual situation. The adjustment principle is: select a region where the fluorescence signal is relatively stable before exponential amplification; avoid signal fluctuations at the beginning of fluorescence acquisition; and reduce the endpoint (Ct) by 1-2 cycles compared to the earliest sample showing exponential amplification. Threshold settings: The principle is to set the threshold line so that it just exceeds the highest point of the normal negative control.

[0079] 3) Result Interpretation

[0080] Table 4

[0081]

[0082] Example 3: Detection results of test samples of the composition of the present invention

[0083] The primers and probes shown in Example 1 were used to perform PCR detection of Marburg virus, Rift Valley fever virus, Chikungunya virus, and Alkuma hemorrhagic fever virus on a Hongshi real-time PCR instrument, following the method in Example 2. The detection results are as follows: Figure 1 As shown in the figure, the compositions of the present invention can effectively detect various pathogens.

[0084] Example 4: Sensitivity of the composition of the present invention

[0085] Using the composition from Example 1 of this invention, LOD (sensitivity) detection was performed on various targets at concentrations of 4000, 1000, 400, and 200 copies / ml to simulate clinical samples. Twenty multiplex PCR tests were performed on a Hongshi real-time PCR instrument. The detection results for the 400 copies / ml sample are as follows: Figures 2-5 As shown, samples with concentrations as low as 400 copies / mL can still be accurately detected in each channel with a detection rate of 100%, indicating that the sensitivity of the composition of the present invention is 400 copies / mL.

[0086] Example 5: Specificity of the composition of the present invention

[0087] The composition of this invention showed no cross-reactivity with common pathogens of hemorrhagic fever and other pathogens with similar infection symptoms (hepatitis A virus, hepatitis B virus, hepatitis C virus, rubella virus, human immunodeficiency virus type 1, Escherichia coli, Pseudomonas aeruginosa, human parainfluenza virus type 3, cytomegalovirus, Coxsackievirus A, Bordetella pertussis, Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Mycobacterium tuberculosis). Results are as follows... Figure 6 As shown, the compositions of the present invention have excellent specificity.

[0088] Example 6: Precision of the composition of the present invention

[0089] Positive samples were diluted to two concentration levels: intermediate positive (R1) and critical positive (R2) (each 1.0 × 10⁻⁶). 5 copies / ml and 1.0×10 3Intra-assay and inter-assay precision were determined using copies / ul, with each sample measured 10 times. Results showed a 100% detection rate for both strong and weak positive reference samples, and the coefficient of variation (CV) of the intra-assay and inter-assay detection Ct values ​​was less than 5%. This indicates that the kit exhibits excellent intra-assay and inter-assay precision. Validation was performed using Marburg virus samples, with the following results. Figure 7 As shown.

[0090] Comparative Example 1: Other primers and probes designed in this invention that do not perform well.

[0091] Due to the principle of complementary base pairing, primers and / or probes can form dimers, but this probability is low and can be eliminated at the initial design stage. However, when detecting multiple pathogens together, there are numerous primers and probes, and dimers can easily form between primers, probes, or between different primers and probes. To ensure the conservation of the design (conservatism is crucial for detection accuracy) while also considering the mutual interference between different primers and probes, careful primer and probe design is required.

[0092] Therefore, the inventors also designed other primers and probes to form different detection systems (sequences not shown), which were also used to detect the aforementioned pathogens. Specific detection results are as follows: Figures 8-11 As shown in the figure, the primers and probes for detecting the four targets performed well in the single-detection system, but the detection was affected in the quadruple-detection system, with obvious amplification curve stratification and a significant decrease in fluorescence increment. Figure 12 As shown, the advantages of the compositions of the present invention are further illustrated.

Claims

1. A composition for detecting hemorrhagic fever infection pathogens, comprising: The upstream primers, downstream primers, and probes for detecting Marburg virus are shown in SEQ ID NO:1-3; For example, the upstream primers, downstream primers and probes for detecting Rift Valley fever virus shown in SEQ ID NO:4-6; For example, the upstream primers, downstream primers, and probes for detecting Chikungunya virus shown in SEQ ID NO:7–9; and The upstream primers, downstream primers, and probes for detecting Alkumar hemorrhagic fever virus are shown in SEQ ID NO:10-12.

2. The composition according to claim 1, characterized in that, The fluorescent groups of the probes in the composition are different from each other and do not interfere with each other.

3. The composition according to claim 1, characterized in that, The composition also includes upstream primers, downstream primers, and probes for detecting the internal standard.

4. The composition according to claim 3, characterized in that, The fluorescent reporter group for the Marburg virus probe is FAM; the fluorescent reporter group for the Rift Valley fever virus probe is ROX; the fluorescent reporter group for the Chikungunya virus probe is CY5; and the fluorescent reporter group for the Alkuma hemorrhagic fever virus probe is HEX.

5. The composition according to any one of claims 1 to 4, characterized in that, The components of the composition exist in a mixed form.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a kit for detecting hemorrhagic fever pathogens, wherein, The pathogens are Marburg virus, Rift Valley fever virus, Chikungunya virus and / or Alkuma hemorrhagic fever virus.

7. A kit for detecting hemorrhagic fever pathogens, said kit comprising the composition as described in any one of claims 1 to 5.

8. The reagent kit according to claim 7, characterized in that, The kit also includes negative and positive controls.

9. The reagent kit according to claim 7 or 8, characterized in that, The kit also includes: nucleic acid release reagent, nucleic acid extraction reagent, reverse transcriptase, DNA polymerase, dNTPs, PCR buffer, and Mg. 2+ At least one of them.

10. Use of a composition for preparing a reagent for detecting hemorrhagic fever infection pathogens, the detection comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition as described in any one of claims 1 to 5 or the kit as described in any one of claims 7 to 9; 3) Obtain and analyze the results.

Images