WO2016082691A1 - Kit for rt-pcr detection of chikungunya and test method thereof - Google Patents

Abstract

Disclosed are a kit for RT-PCR detection of chikungunya and a test method thereof. The kit comprises FRET-PCR standard substances, the upstream primer of the nucleotide sequence of SEQ ID NO: 1, the 6-FAM probe of the nucleotide sequence of SEQ ID NO: 2, the LCRed640 probe of the nucleotide sequence of SEQ ID NO: 3, and the downstream primer of the nucleotide sequence of SEQ ID NO: 4.

Description

Kit for detecting chikungunya virus by reverse transcription PCR and detection method thereof Technical field

The invention relates to the field of biological science and technology, in particular to a kit for detecting chikungunya virus by reverse transcription PCR and a detection method thereof.

Background technique

Chikungunya fever, CHIK is caused by Chikungunya virus, CHIKV, and is mainly transmitted by Aedes aegypti, Aedes albopictus, Aedes africana and other blood-sucking insects. The medium is an acute viral vector infection with fever, rash and severe joint pain as the main clinical symptoms. The incubation period is 3-12 days. It is mainly popular in Africa and Southeast Asia, but in recent years it has been popular in the East African coast, the Indian Ocean islands, the Americas and the Caribbean. The epidemic areas are constantly expanding and the number of cases is increasing. At present, China is mainly a case of imported infection, but some scholars have detected chikungunya virus antibodies from human serum and pigs, mice and other serum and isolated pathogens. It is mainly popular in Africa and Southeast Asia, but in recent years, the prevalence of the region has been expanding, and the number of cases has also increased. Moreover, in October 2010, the first epidemic of the Chikungunya community in China occurred in Dongguan, Guangdong Province. CHIKV belongs to the Togaviridae alphavirus Alphavirus. It can be divided into 4 genotypes by phylogenetic analysis of the viral E1 gene: Chikungunya virus West African genotype, CHIKV-WA, base hole Kenyan virus Asian genotype Chikungunya virus Asian genotype, CHIKV-Asian, Chikungunya virus Indian Ocean genotype Chikungunya virus Indian Ocean genotype, CHIKV-IO, Chikungunya virus East / Central / South African genotype Chikungunya virus East / Central /South African genotype,CHIKV-ECSA. However, in recent years, with the acceleration of the globalization process and the intensification of global warming, conditions have emerged for the spread of the disease from epidemic areas to non-popular areas; at the same time, because Chikungunya fever is similar to the clinical symptoms of dengue fever and malaria. The same media, so many Chikungunya fever was misdiagnosed as dengue fever.

At present, the detection methods of Chikungunya virus mainly include: (1) virus isolation and culture. The virus can be propagated in C6/36, BHK-21, Verona, Hela cells and primary mouse kidney cells, as well as in suckling mice, mosquitoes and other animals. The virus isolation and culture has the advantages of high sensitivity and specificity, but at the same time High requirements for culture environment, personnel, culture conditions and equipment;

(2) Serological methods. Serological methods based on immunochromatography, immunofluorescence and ELISA are the most widely used methods for laboratory diagnosis and commercialization of kits. Serological testing is currently the most widely used method, but antibodies are generally infected in the body. The cross-reaction between Chikungunya virus and Chikungunya virus with different genotypes and other similar pathogens after 5 days makes the detection and diagnosis of Chikungunya fever epidemic difficult. ;

(3) Methods of molecular biology. Reverse transcription PCR, which is a real-time fluorescent RT-PCR-based molecular biology method, provides a rapid and accurate diagnostic method for the diagnosis, prevention and control of Chikungunya virus infection. However, current real-time fluorescent RT-PCR assays are still unable to detect Chikungunya disease in all genotypes in real time, specifically and sensitively.

(1) Virus isolation. CHIKV can proliferate in C6/36, BHK-21, Verona, Hela cells and primary mouse kidney cells, producing typical cytopathic effects and plaques; separating cells with the above cells is as sensitive as separating viruses with suckling mice. . It can be propagated in suckling mice, certain vertebrate cells and mosquitoes. Virus isolation methods include intracerebral inoculation, vertebrate cell culture, and mosquito cell culture. The most common method is cell seed culture.

(2) Serological testing. Serological testing has a wide range of time, both in the detection of antigens in the body and in the detection of antibodies. IgM and IgG antibodies can be detected in the patient's serum 3-6 days after CHIKV infection and clinical signs appear. At present, there are few studies on CHIKV antigen serological detection methods, and there are many antibody detection methods. It mainly includes immunochromatography, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition, and virus neutralization assays.

(a) Immunochromatography. When a sufficient amount of sample is added to the test card sample well, the CHIKV antibody in the sample binds to the CHIKV antigen conjugate and is captured by the anti-antibody coated on the membrane. Commercially available test kits are available. The method is simple in operation, does not require large-scale instruments and equipment, and is suitable for detecting first-line and primary-level medical units to carry out primary selection of the disease, but the sensitivity and specificity of the method are low, and the positive cases of the detection need further diagnosis by other methods.

(b) Immunofluorescence. Also known as the fluorescent antibody method, it is a method of combining antigen and antibody binding reactions with morphology. The method integrates the specificity of serological reaction, the sensitivity of fluorochrome and microscopic examination, and expands the effect of immunological diagnosis. It is an important research method of modern immunology, and there are commercial kits. The method has high sensitivity and is widely used in the field of CHIK epidemic recovery investigation, but it has high requirements on the experimenter's experience, and it is necessary to prevent false positive diagnosis results, and a precision instrument such as a fluorescence microscope is required.

(c) Hemagglutination inhibition test. When the pH and temperature are controlled under appropriate conditions, CHIKV can agglutinate the pigeon's red blood cells, and in the presence of specific antibodies, the agglutination is inhibited due to the combination of the antibody and the viral antigen. The method has the advantages of high specificity, high sensitivity and easy operation, and does not Special equipment is required, but the probability of false positives is also high.

(d) Neutralization test. The method is highly specific and can be tested by antibody cross-reactivity or specific monoclonal antibodies, and can be identified with other viruses of the same group. This method belongs to the CHIK diagnosis test, but requires the CHIKV live virus. The test operation has certain risks. At present, the regulation of CHIKV culture in China needs to be carried out in the BSL-3 laboratory, thus limiting the application of the method.

(e) ELISA method. The IgG antibody in the serum of the case can be detected by indirect IgG ELISA, and the IgG antibody in the serum of the sample can be combined with the CHIKV antigen coated on the ELISA plate, and the sample is judged by reading the OD value after adding the color developer. Negative or positive. The method has a commercial kit, and the operation is simple, and there is no special requirement for the instrument, but the basis of the detection of the method is that the level of the antibody in the serum is required to reach the detection level, generally 5 days after the virus infects the body.

(3) Methods for molecular biological detection. Advances in molecular biology techniques provide fast, reliable techniques for the detection and characterization of pathogens. Molecular biology testing is an ideal early diagnosis method for CHIKV compared to serological testing. The required sample size of the method is extremely small, and accurate results can be obtained in a short time; at the same time, samples of different sources can be detected, and virus culture liquid, blood, and mosquito specimens can be detected by molecular biological methods.

(a) Isothermal gene amplification technology. In this method, four kinds of primers are set for six specific parts of the target gene, and the new strand synthesis is catalyzed by Bst DNA polymerase having strand displacement activity under constant temperature conditions, thereby efficiently amplifying the target gene. The method has high amplification efficiency and simple operation, and does not require large-scale precision instruments in the experiment process, but the sensitivity of the method is low, and it is difficult to select 4 pairs of primers for the conservative path of the more complex pathogens.

(b) RT-PCR is verse Transcription Polymerase Chain Reaction, RT-PCR technology. The PCR method is a sensitive, specific, rapid, and low-pollution detection technique, especially real-time fluorescence RT-CPR. Using routine RT-PCR detection methods, viral nucleic acids can be detected in the serum of patients with multiple infections within 4 days after onset; and more sensitive real-time fluorescent RT-PCR technology can detect viral nucleic acids even after 7 days of onset. . Therefore, for suspicious cases with fever, the preferred laboratory test method is real-time fluorescent RT-PCR, and the PCR product can be sequenced to determine exactly which genotype is infected or infected.

The Chikungunya virus belongs to the Togaviridae alphavirus Alphavirus, and the alphavirus has many members and is classified and complex. So far, it has been divided into seven antigenic complexes. The genome is a non-segmented, positive-stranded RNA containing five structural proteins, capsid C, envelope proteins E1, E2, E3 and 6K and four non-institutional proteins nsP1, nsP2, nsP3 and nsP4. The envelope protein E1 is important for its antigenicity and taxonomy. CHIKV can be divided into 4 genotypes by phylogenetic analysis of the viral E1 gene: Chikungunya virus, West Africa genotype Chikungunya virus West African genotype, CHIKV-WA, mainly in West Africa, the Americas and the Caribbean; Chikungunya virus Asian genotype, CHIKV-Asian, mainly popular in Southeast Asia and South Asia; Chikungunya virus Indian Ocean Genotype Chikungunya virus Indian Ocean genotype, CHIKV-IO, this genotype is a recently popular and defined genotype, mainly prevalent in the Indian Ocean islands and India; Chikungunya virus east/middle/South African genotype Chikungunya virus East/Central/ South African genotype, CHIKV-ECSA, is the most prevalent of the four genotypes of CHIKV, mainly distributed in the eastern, central and southern parts of Africa and the Indian Ocean. Chikungunya was first discovered in Tanzania and is mainly found in tropical and subtropical regions of Africa and Asia. However, in recent years, with the acceleration of globalization and the intensification of global warming, conditions have emerged for the spread of the disease from epidemic areas to non-popular areas. In the southern coastal provinces of China, there are imported cases of Chikungunya fever every year. At the same time, the coastal defense line of China is threatened. At the same time, because Chikungunya fever is similar to the clinical symptoms of dengue fever and malaria and the media is similar, many Chikungunya fever are misdiagnosed as dengue virus infection.

Therefore, the rapid and accurate detection and detection method of Chikungunya virus is helpful to understand the biological characteristics and clinical diagnosis of Chikungunya virus, and apply it to the detection of Chikungunya virus infection to prevent it. The introduction of China is of great significance.

At present, there is a lack of a highly sensitive, specific, and rapid detection PCR kit for detecting Chikungunya virus and its detection method.

Summary of the invention

The object of the present invention is to provide a highly sensitive, specific and rapid detection kit for detecting Chikungunya virus by reverse transcription PCR and a detection method thereof.

The technical scheme of the present invention is as follows: The present invention provides a kit for detecting chikungunya virus by reverse transcription PCR, which comprises primers, probes and FRET-PCR standards,

The primers are an upstream primer and a downstream primer;

The probe is a 6-FAM probe and a LCRed 640 probe;

The upstream primer has the nucleotide sequence of SEQ ID No. 1;

The 6-FAM probe has the nucleotide sequence of SEQ ID No. 2;

The LCRed640 probe has the nucleotide sequence of SEQ ID No. 3;

The downstream primer has the nucleotide sequence of SEQ ID No. 4.

Further, the kit comprises: a primer, a 6-FAM probe, a LCRed640 probe, a PCR buffer, a hot start Taq enzyme, a dNTP, a FRET-PCR standard, and a PCR negative control; the PCR negative control is double steaming water.

Further, the FRET-PCR standard is a primer and probe pair DNA of the FRET-PCR The amplified plasmid fragment obtained by amplification of the plasmid standard template was inserted into a recombinant plasmid constructed from the pUC57 vector.

Further, the concentration of the DNA plasmid standard template is 100 gene copies/μl.

The real-time fluorescence quantitative FRET-PCR amplification detection system of the kit for detecting chikungunya virus by reverse transcription PCR of the invention comprises real-time fluorescence quantitative FRET-PCR amplification target comprising a DNA plasmid standard template and a PCR negative control;

The real-time fluorescence quantitative FRET-PCR amplification detection system includes 20 μl of amplification system: 10 μl of sample DNA template or quantitative DNA standard reagent, 1×PCR buffer, 1 μM upstream primer, 1 μM downstream primer, 0.2 μM 6-FAM probe, 0.2 μM LCRed640 probe, 2 units of commercial Taq enzyme, 200 μM dNTP.

The invention provides a reverse transcription PCR amplification detection system for reverse transcription PCR detection of Chikungunya virus kit, and the reverse transcription PCR amplification target comprises an RNA standard template, a negative control for RNA extraction and RT- PCR negative control;

The reverse transcription PCR amplification assay system includes 20 μl of amplification system containing: 10 μl of sample RNA template or quantitative RNA standard reagent, 1×PCR buffer, 1 μM upstream primer, 1 μM downstream primer, 0.2 μM 6-FAM probe, 0.2 μM LCRed640 probe, 2 units of commercial Taq enzyme, 200 μM dNTP, 0.14 units of commercial reverse transcriptase, 20 units of commercial RNase inhibitor.

The invention discloses a method for detecting a chikungunya virus kit by reverse transcription PCR, wherein the PCR amplification setting reaction conditions include: pre-denaturation, 18 high-rigidity cycles with decreasing temperature, and 40 under-rigid fluorescence Obtain a cycle, a continuous fluorescence-obtained melting cycle and a cooling cycle; pre-denaturation: 1x2min@95°C; 18 high-rigid cycles with decreasing temperature: 6x1sec@95°C, 12sec@70°C, 8sec@72°C; 9x1sec @95°C, 12sec@68°C, 8sec@72°C; 3x1sec@95°C, 12sec@66°C, 8sec@72°C; 40 less stringent fluorescence acquisition cycles: 40x1sec@95°C, 8sec@56°C, 30sec@ 67 ° C, and 30 sec @ 72 ° C; 1 melting cycle: 1 x 1 sec @ 95 ° C, 10 sec @ 38 ° C, @ 85 ° C continuous collection of fluorescence; cooling cycle: 1 x 1 sec @ 38 ° C.

A method for detecting a chikungunya virus kit for reverse transcription PCR according to the present invention, wherein the reverse transcription PCR amplification set reaction conditions include: reverse transcription, pre-denaturation, and 18 temperature-decreasing High stringency cycle, 40 less stringent fluorescence acquisition cycles and 1 cooling cycle;

Reverse transcription: 1x30min@55°C; pre-denaturation: 1x2min@95°C; 18 high rigorous cycles of temperature decrease: 6x1sec@95°C, 12sec@70°C, 8sec@72°C; 9x1sec@95°C, 12sec@68°C , 8sec@72°C; 3x1sec@95°C, 12sec@66°C, 8sec@72°C; 40 less stringent fluorescence acquisition cycles: 40x1sec@95°C, 8sec@56°C, 30sec@67°C, and30sec@72°C; Cooling cycle: 1x1sec@38°C.

The kit for detecting chikungunya virus by reverse transcription PCR according to the present invention is applied to chikungunya fever.

The beneficial effects: the reverse transcription PCR system established by the invention can effectively amplify RNA, and can rapidly and efficiently amplify chikungunya virus RNA nucleic acid in clinical samples, which is rapid when the Chikungunya fever bursts. Accurate diagnosis of large numbers of clinical samples and providing a solid basis for controlling the disease as quickly as possible. The reverse transcription PCR system can specifically and stably amplify Chikungunya virus of all genotypes. The invention also has a method for highly sensitive, specific and rapid detection of all chikungunya virus genotypes CHIKV-Asian, CHIKV-ECSA, CHIKV-IO, CHIKV-WA.

The invention has the following advantages:

(1) The present invention can specifically detect the genotype of all chikungunya viruses. The RT-PCR system of the invention has extremely high sensitivity. In the early or recovery phase of viral infection, the virus content in the body is relatively low, which requires sensitive and accurate detection methods, so that infectious diseases can be diagnosed, monitored and controlled in a timely and rapid manner, especially like Chikungun. Ya fever is an acute infectious disease. Conventional RT-PCR detection methods generally detect viral nucleic acid in the serum of most patients within 4 days after onset; and more sensitive real-time fluorescent RT-PCR technology can detect viral nucleic acid even after 7 days of onset. Therefore, the preferred laboratory test method for suspicious CHIK infection in the febrile phase is real-time fluorescent RT-PCR, and the reverse transcription PCR product can be used to determine the nucleic acid sequence and accurately determine whether it is CHIKV infection. The real-time fluorescent RT-PCR system of the invention can detect a single copy of the plasmid nucleic acid, a plasmid standard carrying the four genotype DNA nucleic acids of Chikungunya virus, and can effectively amplify the minimum 10-7 avian influenza virus H1N1. RNA nucleic acid, an RNA nucleic acid standard prepared from chicken embryo allantoic fluid.

(2) The invention is convenient to operate and is suitable for detecting a large number of samples. Chikungunya is currently a local epidemic, mainly in Africa, Southeast Asia, the Mediterranean and the Caribbean, but as the globalization process accelerates and global warming worsens, the disease is spread from endemic areas. The spread of non-popular areas creates conditions, which urgently require a fast and accurate detection method to be better applied to the border inspection, providing a diagnostic basis for preventing the introduction of the disease; at the same time, Chikungunya as a An acute infectious disease is particularly important for prevention in endemic areas, especially in areas where outbreaks occur. (3) The present invention establishes a reverse transcription PCR system capable of detecting all chikungunya virus genotypes according to the conserved interval in the four genotype nucleic acid sequences of chikungunya virus. First, obtain the full-length sequence of all Chikungunya virus genotypes and other related pathogens with high homology from NCBI, and select relatively conservative intervals to design primers and probes, and then use the primers and probe pairs. Samples are tested in real time, specifically, sensitively and quickly.

(4) The present invention ensures that the system does not amplify other microorganisms other than Chikungunya virus, especially other pathogens having similar homology, such as Mayaro virus, MAYV, Gatah virus, GETV, O'nyong-nyong virus, ONNV, Simon's Forest Virus Semliki Forest virus, SFV, Venezuelan equine encephalitis virus, VEEV, Eastern equine encephalitis virus, EEEV and Sindbis virus, SINV; and infections with similar clinical manifestations and the same media, often misdiagnosed Diseases, such as dengue fever, malaria, etc. The present invention selects a conserved region of chikungunya virus as a target fragment to design primers and probes. The general idea is: subtly design primers and probes for RT-PCR to specifically amplify all chikonia virus genotypes, such as Chikungunya virus Asian genotype CHIKV-Asian, Chikungunya The virus East/Middle/South African genotype CHIKV-ECSA, Chikungunya virus Indian Ocean genotype CHIKV-IO, Chikungunya virus West African genotype CHIKV-WA, to quickly determine a positive sample.

DRAWINGS

Figure 1 is a schematic diagram of an upstream primer of reverse transcription PCR of the present invention;

Figure 2 is a schematic diagram of a 6-FAM probe of reverse transcription PCR of the present invention;

Figure 3 is a schematic illustration of the LCRed640 probe of the reverse transcription PCR of the present invention;

Figure 4 is a schematic diagram of a downstream primer of reverse transcription PCR of the present invention;

Figure 5 is a schematic diagram showing the amplification curve of the chikungunya virus Asian genotype of the present invention;

Figure 6 is a schematic view showing the melting curve of the chikungunya virus Asian genotype of the present invention;

Figure 7 is a schematic diagram showing the amplification curve of the Chikungunya virus ECSA genotype of the present invention;

Figure 8 is a schematic view showing the melting curve of the Chikungunya virus ECSA genotype of the present invention;

Figure 9 is a schematic diagram showing the amplification curve of the Chikungunya virus IO genotype of the present invention;

Figure 10 is a schematic view showing the melting curve of the Chikungunya virus IO genotype of the present invention;

Figure 11 is a schematic diagram showing the amplification curve of the chikungunya virus WA genotype of the present invention;

Figure 12 is a schematic view showing the melting curve of the chikungunya virus WA genotype of the present invention;

Figure 13 is a schematic diagram showing the amplification curves of four chikungunya virus genotype plasmids of the present invention;

Figure 14 is a schematic view showing the melting curves of four chikungunya virus genotype plasmids of the present invention;

Figure 15 is a schematic diagram showing the amplification curve of the reverse transcription PCR system of the present invention;

Figure 16 is a schematic view showing the melting curve of the reverse transcription PCR system of the present invention;

Figure 17 is a schematic diagram showing the agarose gel electrophoresis of four chikungunya virus genotypes of the present invention.

detailed description

The invention will be further described in detail with reference to the accompanying drawings and specific embodiments, which are not to be construed as limiting the scope of the invention.

Example 1

The invention provides a kit for detecting chikungunya virus by reverse transcription PCR, comprising primers, probes and FRET-PCR standards, wherein the primers are upstream primers and downstream primers; the probe is 6-FAM Probe and LCRed640 probe;

1 to 4, Fig. 1 is a schematic diagram of an upstream primer of reverse transcription PCR of the present invention; a reverse transcription PCR upstream primer sequence: 5'-CGGCTTCTTCAATATGATGCAGATG-3' (25 bp). This primer sequence is fully aligned with the chikungunya nucleic acid sequence of all genotypes.

2 is a schematic diagram of a 6-FAM probe of reverse transcription PCR of the present invention; 6-FAM probe sequence of reverse transcription PCR: 5'-GACACAATGGCAGTCACAGGCAGT-6-FAM-3' (24 bp), this sequence is in the figure A symmetrical strand nucleotide sequence of the sequence is obtained. This FAM probe sequence has a mismatch base with the CHIKV-IO genotype nucleic acid sequence, and is in complete agreement with the chikungunya virus nucleic acid sequences of other genotypes.

Figure 3 is a schematic diagram of the LCRed640 probe of the reverse transcription PCR of the present invention; LCRed640 probe sequence of reverse transcription PCR: 5'-LCRed640-TACACCGCCTGGARATACTTTTGTGGT-phosphate group-3' (27 bp), this sequence is the sequence obtained in the figure Symmetrical chain nucleotide sequence. The downstream primer sequence has one merging base R, and the merging base R can simultaneously amplify the A and G bases. Therefore, the LCRed640 probe sequence has a mismatch base with the CHIKV-WA genotype nucleic acid sequence, and is completely coincident with the chikonia virus nucleic acid sequences of other genotypes.

Figure 4 is a schematic representation of a downstream primer for reverse transcription PCR of the present invention; a reverse transcription PCR downstream primer sequence: 5'-GCATTTTGCCTTCGTAATGCAACGA-3' (25 bp), which is a symmetrical strand nucleotide sequence of the sequence obtained in the figure. This primer sequence is fully aligned with the Chikungunya virus nucleic acid sequences of all genotypes.

The nucleotide sequences of the primers and probes used in the Chikungunya virus reverse transcription PCR assay are as follows:

Upstream primer: 5'-CGGCTTCTTCAATATGATGCAGATG-3'SEQ ID No. 1;

6-FAM probe: 5'-GACACAATGGCAGTCACAGGCAGT-6-FAM-3'SEQ ID No. 2;

LCRed640 probe: 5'-LCRed640-TACACCGCCTGGARATACTTTTGTGGT-phosphate group-3'SEQ ID No. 3;

Downstream primer: 5'-GCATTTTGCCTTCGTAATGCAACGA-3'SEQ ID No. 4.

The kit includes: a primer, a 6-FAM probe, a LCRed640 probe, a PCR buffer, a hot start Taq enzyme, a dNTP, a FRET-PCR standard, and a PCR negative control; the PCR negative control is double distilled water.

The FRET-PCR standard is a recombinant plasmid constructed by inserting an amplified nucleotide fragment obtained by amplification of a DNA plasmid standard template with a primer and a probe of the FRET-PCR into a pUC57 vector.

The concentration of the DNA plasmid standard template was 100 gene copies/μl.

The real-time fluorescence quantitative FRET-PCR amplification detection system of the kit for detecting chikungunya virus by reverse transcription PCR of the invention comprises real-time fluorescence quantitative FRET-PCR amplification target comprising a DNA plasmid standard template and a PCR negative control;

The real-time fluorescence quantitative FRET-PCR amplification detection system includes 20 μl of amplification system: 10 μl of sample DNA template or quantitative DNA standard reagent, 1×PCR buffer, 1 μM upstream primer, 1 μM downstream primer, 0.2 μM 6-FAM probe, 0.2 μM LCRed640 probe, 2 units of commercial Taq enzyme, 200 μM dNTP.

The invention provides a reverse transcription PCR amplification detection system for reverse transcription PCR detection of Chikungunya virus kit, and the reverse transcription PCR amplification target comprises an RNA standard template, a negative control for RNA extraction and RT- PCR negative control;

The real-time fluorescence quantitative FRET-PCR amplification detection system includes 20 μl of amplification system: 10 μl of sample DNA template or quantitative DNA standard reagent, 1×PCR buffer, 1 μM upstream primer, 1 μM downstream primer, 0.2 μM 6-FAM probe, 0.2 μM LCRed640 probe, 2 units of commercial Taq enzyme, 200 μM dNTP.

The invention provides a PCR amplification system for a kit for detecting chikungunya virus by reverse transcription PCR, and the reverse transcription PCR amplification target comprises an RNA standard template, a negative control for RNA extraction and a negative RT-PCR. Control

The reverse transcription PCR amplification detection system comprises 20 μl of amplification system comprising: 10 μl of sample RNA template or quantitative RNA standard reagent, 1×PCR buffer, 1 μM upstream primer, 1 μM downstream primer, 0.2 μM 6-FAM probe, 0.2 μM LCRed640 probe, 2 units of commercial Taq enzyme, 200 μM dNTP, 0.14 units of commercial reverse transcriptase, 20 units of commercial RNase inhibitor.

The invention discloses a method for detecting a chikungunya virus kit by reverse transcription PCR, wherein the PCR amplification setting reaction conditions include: pre-denaturation, 18 high-rigidity cycles with decreasing temperature, and 40 under-rigid fluorescence Obtain a cycle, a continuous fluorescence-obtained melting cycle and a cooling cycle; pre-denaturation: 1x2min@95°C; 18 high-rigid cycles with decreasing temperature: 6x1sec@95°C, 12sec@70°C, 8sec@72°C; 9x1sec @95°C, 12sec@68°C, 8sec@72°C; 3x1sec@95°C, 12sec@66°C, 8sec@72°C; 40 less stringent fluorescence acquisition cycles: 40x1sec@95°C, 8sec@56°C, 30sec@ 67 ° C, and 30 sec @ 72 ° C; 1 melting cycle: 1 x 1 sec @ 95 ° C, 10 sec @ 38 ° C, @ 85 ° C continuous collection of fluorescence; cooling cycle: 1 x 1 sec @ 38 ° C.

A method for detecting a chikungunya virus kit for reverse transcription PCR according to the present invention, wherein the reverse transcription PCR amplification set reaction conditions include: reverse transcription, pre-denaturation, and 18 temperature-decreasing High stringency cycle, 40 less stringent fluorescence acquisition cycles and 1 cooling cycle;

Reverse transcription: 1x30min@55°C; pre-denaturation: 1x2min@95°C; 18 high rigorous cycles of temperature decrease: 6x1sec@95°C, 12sec@70°C, 8sec@72°C; 9x1sec@95°C, 12sec@68°C , 8sec@72°C; 3x1sec@95°C, 12sec@66°C, 8sec@72°C; 40 less stringent fluorescence acquisition cycles: 40x1sec@95°C, 8sec@56°C, 30sec@67°C, and30sec@72°C; Cooling cycle: 1x1sec@38 °C.

The kit for detecting chikungunya virus by reverse transcription PCR according to the present invention is applied to chikungunya fever.

After obtaining the following four genotypes of Chikungunya virus and other gene sequences of other homologous species from GenBank (www.ncbi.nlm.nih.gov), use the Clustal Multiple Alignment Algorithm method for all The sequences are aligned.

Figure 5 is a schematic diagram showing the amplification curve of the chikungunya virus Asian genotype of the present invention; indicating that the system of the present invention can detect a single copy of the chikungunya virus Asian genotype DNA molecule in the reaction system, and has reproducibility;

Figure 6 is a schematic diagram showing the melting curve of the chikungunya virus Asian genotype of the present invention; the system of the present invention has a stable Tm value for the chikungunya virus Asian genotype.

Figure 7 is a schematic diagram showing the amplification curve of the chikungunya virus ECSA genotype of the present invention; indicating that the system of the present invention can detect a single copy of the chikungunya virus ECSA genotype DNA molecule in the reaction system, and is reproducible;

Figure 8 is a schematic representation of the melting curve of the Chikungunya virus ECSA genotype of the present invention; it is shown that the system of the invention has a stable Tm value for the Chikungunya virus ECSA genotype.

Figure 9 is a schematic diagram showing the amplification curve of the chikungunya virus IO genotype of the present invention; indicating that the system of the present invention can detect a single copy of the chikungunya virus IO genotype DNA molecule in the reaction system, and has reproducibility;

Figure 10 is a schematic representation of the melting curve of the chikungunya virus IO genotype of the present invention; it is shown that the system of the invention has a stable Tm value for the chikungunya virus IO genotype.

Figure 11 is a schematic diagram showing the amplification curve of the chikungunya virus WA genotype of the present invention; indicating that the system of the present invention can detect a single copy of the chikungunya virus WA genotype DNA molecule in the reaction system, and is reproducible;

Figure 12 is a schematic representation of the melting curve of the chikungunya virus WA genotype of the present invention; it is shown that the system of the invention has a stable Tm value for the chikungunya virus WA genotype.

Figure 13 is a schematic diagram showing the amplification curves of four chikungunya virus genotype plasmids of the present invention; showing that the system of the present invention can detect a single copy of chikungunya virus Asian, ECSA, IO and WA genotype DNA in the reaction system. Molecules, and are reproducible;

Figure 14 is a schematic representation of the melting curves of four chikungunya virus genotype plasmids of the invention; showing that the system of the invention has stable Tm values for the Chikungunya virus Asian, ECSA, IO and WA genotypes.

As shown in FIG. 5 to FIG. 9, the present invention detects four plasmids carrying the DNA sequences of four chimeric Kenyan virus genotypes (CHIKV-Asian, CHIKV-ECSA, CHIKV-IO, CHIKV-WA) (Jinsley) Biotechnology, China Nanjing) Standards. The results show that the system of the present invention can specifically and efficiently amplify Chikungunya virus DNA of all serotypes;

As shown in Fig. 6 to Fig. 14, the present invention skillfully designs a pair of primers and probes according to the conserved interval of each genotype of chikungunya virus, and can simultaneously detect all four chikungunya virus genotypes (CIKV- Asian, CHIKV-ECSA, CHIKV-IO, CHIKV-WA)

Figure 15 is a schematic diagram showing the amplification curve of the reverse transcription PCR system of the present invention; the influenza virus (H1N1) RNA extracted from the chicken embryo allantoic fluid is subjected to gradient dilution, and the RT-PCR reaction conditions and influenza virus primers of the present invention are used. And probe detection. The results indicate that the reverse transcription PCR system of the present invention can detect a minimum of 10-7 copies of the RNA of interest and is reproducible;

Figure 16 is a schematic illustration of the melting curve of the reverse transcription PCR system of the present invention; all dilution gradients exhibit a stable melting curve.

Figure 17 is a schematic diagram showing the agarose gel electrophoresis of four chikungunya virus genotypes of the present invention. The target nucleotide fragment size of the four chikungunya virus genotypes (Asian, ECSA, IO and WA) amplified by the RT-PCR system of the present invention is 139 base pairs (bp). The lanes are: Lane-1: Standard (Ladder), Lane-2: Chikungunya virus Asian genotype (CHIKV-Asian), Lane-3: Chikungunya virus East/Middle /Chikungunya virus East/Central/South African genotype (CHIKV-ECSA), Lane-4: Chikungunya virus West African genotype CHIKV-WA, Lane-5: Chikungun Chikungunya virus Indian Ocean genotype (CHIKV-IO), Lane-6: Negative control (NC). Among them, the standard strips are 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp and 300 bp. The fluorescence amplification curve of the positive sample and the positive control PCR showed the appearance or enhancement of fluorescence at 640 nm, and the band size of the PCR product in agarose gel electrophoresis was 139 bp.

Determination of the specificity of the reverse transcription PCR of the present invention:

The specificity of the invention is guaranteed in three ways:

(1) Primers and probes designed by GenBank BLAST search, it was confirmed that the primers designed by the present invention can specifically amplify all chikungunya virus genotypes (CHIKV-Asian, CHIKV-ECSA, CHIKV-IO, CHIKV- WA), without amplifying other non-Chikungunya fever pathogens, especially with similar homology (Mayaro virus, Gaeta virus, Anion, Anthony forest virus, Venezuelan equine encephalitis, Oriental horse) Pathogen nucleic acid of encephalitis and Sindbis virus, etc. or clinical features (dengue, etc.). It is confirmed by BLAST that the probes and primers of the RT-PCR of the present invention can specifically amplify and detect all the nucleic acids of chikungunya virus but not the nucleic acids of other related viruses;

(2) As shown in Figures 6 to 14, the present invention detects four genotypes of chikungunya virus (CHIKV-Asian, CHIKV-ECSA, CHIKV-IO, CHIKV-WA) Four plasmids (Jinsru Biotechnology, Nanjing, China) standard for DNA sequences. The results show that the system of the present invention can specifically and efficiently amplify Chikungunya virus DNA of all serotypes;

(3) As shown in Fig. 6 to Fig. 14, as shown in Fig. 17, the change in fluorescence intensity (640 nm) of the above amplified subject during PCR and the size of the band of the PCR amplification product in agarose gel electrophoresis were observed. The results showed that the fluorescence curve of the PCR amplification curve of all Chikungunya plasmid nucleic acids appeared or enhanced at a wavelength of 640 nm; while the PCR amplification product showed 139 base pairs (bp) in agarose gel electrophoresis. Strip size

Determination of sensitivity of chikungunya virus by RT-PCR of the present invention:

As shown in Fig. 6 to Fig. 14, four genotypes of Chikungunya virus (CHIKV-Asian, CHIKV-ECSA, CHIKV-IO, CHIKV-WA) were synthesized by Kingsray (Kinsley Biotechnology, Nanjing, China). The DNA sequence of the fragment of interest. The absolute number of copies of the gene contained in the composition is calculated based on the molecular weight and absolute weight of the composition. Subsequently, the composition was diluted to prepare a 10,000-copy, 1,000-copy, 100-copy, 10-copy, 1-copy gene for each 10 μl of the composition. The sensitivity of the present invention for detecting this gene is determined by amplifying a dilution containing different gene concentrations using the system of the present invention. The results show that this invention can amplify a single copy of the gene of interest in the reaction system.

Determination of the efficiency of the reverse transcription PCR system in the present invention:

As shown in Fig. 15 and Fig. 16, chicken embryos were harvested after infection with chicken avian influenza virus H1N1, and RNA was extracted from allantoic fluid using a commercial RNA extraction kit (High Pure RNA Isolation Kit, Roche, Germany). Then, it was subjected to gradient dilution, and 10-3-10-8 was selected for reverse transcription PCR amplification. The results indicate that the reverse transcription PCR system of the present invention can detect a minimum of 10-7 copies of avian influenza virus RNA molecules in the reaction system.

Prepare standard quantitation reagents for PCR:

(1) Preparation of DNA plasmid standards

The DNA sequence of the target fragments of Chikungunya virus four genotypes (CHIKV-Asian, CHIKV-ECSA, CHIKV-IO, CHIKV-WA) was synthesized by Kingsray (Kinsley Biotechnology, Nanjing, China). The absolute number of copies of the gene contained in the composition is calculated based on the molecular weight and absolute weight of the composition. Subsequently, the composition was diluted to prepare a 10,000 copies, 1,000 copies, 100 copies, 10 copies, and 1 copy of the target gene per 10 μl of the composition as a standard. In the kit of the present invention, four genotypes of chikungunya virus and a plasmid standard having a concentration of 104/μl were provided.

(2) Preparation of RNA standards

After infecting chicken embryos with avian influenza virus H1N1, the allantoic fluid was collected, and RNA nucleic acid extraction was performed using a commercial RNA extraction kit (High Pure RNA Isolation Kit, Roche, Germany), followed by TE buffer, pH 8.0 for 10-3. , 10-4, 10-5, 10-6, 10-7, 10-8 gradient dilution as a reverse transcription PCR system A standard of efficiency.

Agarose gel electrophoresis:

Prepare a 3% agarose gel, take 10 μl of PCR amplification product, and use

Safe DNA Gel Stain(

Safe DNA Gel Stain, invitrogen TM by life

US) staining, observed under UV light, can see the target band at 139 bp (Figure 11). The standard used was a 20 bp DNA Ladder (Thermo Scientific O'RangeRuler 20 bp DNA Ladder, ready-to-use,

By Thermo

US)], and its strip size is 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp, 300 bp.

Determination of PCR results:

As shown in Fig. 6 to Fig. 14, the real-time fluorescent PCR amplification target includes a DNA plasmid standard template, a PCR negative control (double distilled water), and a reverse transcription PCR amplification target includes an RNA standard template, a negative control for RNA extraction, and RT-PCR negative control (double distilled water). This invention is highly specific and can detect nucleic acids of all Chikungunya virus genotypes (CHIKV-Asian, CHIKV-ECSA, CHIKV-IO, CHIKV-WA).

Example 2

Transcription method to verify the PCR system of the invention:

Plasmid batch pUC57 containing the Chikungunya virus Asian genotype CHIKV-Asian fragment DNA and T7 promoter was synthesized by Kingsray (Kinsley Biotechnology, Nanjing, China) with appropriate restriction enzymes (Sac I) , Bao Bio, Dalian) to digest the plasmid; increase the concentration of the target DNA by PCR amplification; use a commercial transcription kit (

Kit,

By life

In the United States, the PCR amplification product is transcribed and an RNA product is obtained; after the transcription is completed, the transcript is diluted and used for reverse transcription PCR for amplification.

(1) dilution of the plasmid

The microcentrifuge tube containing the synthetic plasmid was centrifuged at 4000 rpm for 2 minutes, then 40 μl of TE buffer was added, and the solution having a plasmid concentration of 100 ng/μl was prepared at a pH of 8.0;

(2) Enzyme digestion of the plasmid

The plasmid was digested with a commercial restriction endonuclease SacI (Sac I, Bao Bio, Dalian), and the reaction system was 20 μl, and the composition of each reagent was as follows; the reaction liquids were mixed and placed in a 37 ° C environment. Incubating for 1 hour; then, the reaction system mixture was heated in a 56 ° C environment for 20 minutes to cause a restriction enzyme to ignite, thereby terminating the digestion reaction;

Sac I	1μl
10×L Buffer	2μl
DNA (plasmid)	5μl
Sterilized water	12μl
reaction system	20μl

(3) PCR amplification of the product

A fragment containing a T7 transcriptional promoter and a DNA fragment of interest of 733 base pairs (bp) was amplified from the linear plasmid with the following primers to provide sufficient concentration of the target DNA for subsequent transcription, 1 μg/μl or more;

P-upstream primer: 5'-GGTACCTCGCGAATGCATC-3'

P-downstream primer: 5'-CAGGAAACAGCTATGACCA-3'

(4) In vitro transcription

Dissolve all relevant reagents in the kit at room temperature (take the RNA Polymerase Enzyme Mix in a -20 ° C freezer for use);

Mix the reagents in the table below at room temperature and mix by pipetting up and down;

Amount	Component
7μl	Nuclease-free Water
2μl	ATP solution
2μl	CTP solution
2μl	GTP solution

Incubate for 4 hours at 37 ° C; add 1 μl of TURBO DNase, mix and incubate for 15 minutes at 37 ° C to remove the nucleic acid DNA in the reaction system; and use the above transcription product in TE buffer (pH 8.0) for 10 -3, 10-4, 10-5 dilution; transcript dilution (nucleic acid RNA) obtained by the above method was amplified using the RT-PCR system established in the present invention which can detect all chikungunya virus genotypes.

The results showed that the fluorescence amplification curve of the transcript showed the appearance and enhancement of fluorescence at 640 nm, and there was a single, stable melting peak and Tm value at the melting curve. Therefore, the reverse transcription PCR system established by the invention can effectively amplify RNA, and can rapidly and efficiently amplify chikungunya virus RNA nucleic acid in clinical samples.

The basic principles, main features, and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited by the foregoing embodiments, and that the present invention is only described in the foregoing description and the description of the present invention, without departing from the spirit and scope of the invention. The scope of the invention is defined by the appended claims, the description and the equivalents thereof.

Claims (9)

1. A kit for detecting chikungunya virus by reverse transcription PCR, comprising primers, probes and FRET-PCR standards, characterized in that the primers are upstream primers and downstream primers; the probe is 6-FAM Probe and LCRed640 probe;

   The upstream primer has the nucleotide sequence of SEQ ID No. 1;

   The 6-FAM probe has the nucleotide sequence of SEQ ID No. 2;

   The LCRed640 probe has the nucleotide sequence of SEQ ID No. 3;

   The downstream primer has the nucleotide sequence of SEQ ID No. 4.
2. The kit for detecting chikungunya virus by reverse transcription PCR according to claim 1, wherein the kit comprises: a primer, a 6-FAM probe, a LCRed640 probe, a PCR buffer, a hot start Taq Enzyme, dNTP, FRET-PCR standard, PCR negative control; the PCR negative control is double distilled water.
3. The kit for detecting chikungunya virus by reverse transcription PCR according to claim 2, wherein the FRET-PCR standard is a primer and probe pair DNA plasmid standard of the FRET-PCR The amplified nucleotide fragment obtained by amplification of the template was inserted into a recombinant plasmid constructed from the pUC57 vector.
4. The kit for detecting chikungunya virus by reverse transcription PCR according to claim 3, wherein the concentration of the DNA plasmid standard template is 100 gene copies/μl.
5. A real-time fluorescence quantitative FRET-PCR amplification detection system for reverse transcription PCR detection of Chikungunya virus kit, characterized in that real-time fluorescence quantitative FRET-PCR amplification target includes DNA plasmid standard template, PCR negative control ;

   The real-time fluorescence quantitative FRET-PCR amplification detection system includes 20 μl of amplification system: 10 μl of sample DNA template or quantitative DNA standard reagent, 1×PCR buffer, 1 μM upstream primer, 1 μM downstream primer, 0.2 μM 6-FAM probe, 0.2 μM LCRed640 probe, 2 units of commercial Taq enzyme, 200 μM dNTP.
6. A reverse transcription PCR amplification detection system for reverse transcription PCR detection of Chikungunya virus kit, characterized in that: reverse transcription PCR amplification includes RNA standard template, negative control of RNA extraction and RT- PCR negative control;

   The reverse transcription PCR amplification assay system includes 20 μl of amplification system containing: 10 μl of sample RNA template or quantitative RNA standard reagent, 1×PCR buffer, 1 μM upstream primer, 1 μM downstream primer, 0.2 μM 6-FAM probe, 0.2 μM LCRed640 probe, 2 units of commercial Taq enzyme, 200 μM dNTP, 0.14 units of commercial reverse transcriptase, 20 units of commercial RNase inhibitor.
7. A method for detecting a chikungunya virus by reverse transcription PCR, characterized in that: the PCR amplification setting reaction conditions include: pre-denaturation, 18 high-rigid cycles of temperature decrease, and 40 less stringent Fluorescence obtained cycle, 1 continuous fluorescence obtained melting cycle and 1 cooling cycle; pre-denaturation: 1x 2min@95°C; 18 high-rigid cycles with decreasing temperature: 6x 1sec@95°C, 12sec@70°C, 8sec@72 °C; 9x 1sec@95°C, 12sec@68°C, 8sec@72°C; 3x 1sec@95°C, 12sec@66°C, 8sec@72°C; 40 less stringent fluorescence acquisition cycles: 40x 1sec@95°C, 8sec @56°C, 30sec@67°C, and 30sec@72°C; 1 melting cycle: 1x 1sec@95°C, 10sec@38°C, @85°C continuous fluorescence collection; cooling cycle: 1x 1sec@38°C.
8. A method for detecting a kit for detecting chikungunya virus by reverse transcription PCR, characterized in that: the reverse transcription PCR amplification setting reaction conditions include: reverse transcription, pre-denaturation, and 18 temperature decrement High stringency cycle, 40 less stringent fluorescence acquisition cycles and 1 cooling cycle;

   Reverse transcription: 1x 30min@55°C; pre-denaturation: 1x 2min@95°C; 18 high rigorous cycles of temperature decrease: 6x 1sec@95°C, 12sec@70°C, 8sec@72°C; 9x 1sec@95°C, 12sec@68°C, 8sec@72°C; 3x 1sec@95°C, 12sec@66°C, 8sec@72°C; 40 less stringent fluorescence acquisition cycles: 40x 1sec@95°C, 8sec@56°C, 30sec@67°C , and 30sec@72°C; cooling cycle: 1x 1sec@38°C.
9. Use of the kit for detecting reverse chitokin virus by reverse transcription PCR according to any one of claims 1 to 4 in Chikungunya fever.

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