CN105936943B - A Rapid and Sensitive RT-LAMP Detection Method for Marburg Virus - Google Patents

Abstract

The present invention relates to a kind of Marburg virus RT-LAMP detection methods of rapid sensitive.Detection method of the invention is by design specific primer, isothermal duplication (LAMP) technology mediated by ring, using the visual reagent of addition fluorescence, can be simple and quick detect amplified production；Detection method includes: that sample RNA is extracted, RT-LAMP amplification, as a result interpretation.The present invention can carry out the detection of rapid sensitive, instrument and equipment and easy to operate, detection time section to Marburg virus.The detection efficiency of first-line inspection and quarantine personnel at import and export ports can be increased substantially through the invention, not only workload can have been reduced but also can have solved the problems, such as traditional detection method positive missing inspection that may be present to the maximum extent, to prevent the incoming of Marburg virus to the maximum extent.

Description

A Rapid and Sensitive RT-LAMP Detection Method for Marburg Virus

technical field

The invention relates to the technical field of in vitro diagnostic reagents, in particular to a rapid and sensitive Marburg virus RT-LAMP detection method.

Background technique

With global economic integration and trade liberalization, foreign medical vectors can quickly spread infectious diseases from one country or region to the world through advanced means of transportation and international trade, resulting in international spread. In recent years, new viral infectious diseases have frequently broken out in countries all over the world, posing a serious threat to human health and causing huge economic losses to society. For example, the Severe Acute Respiratory Syndrome (SARS) epidemic in some countries and regions in 2003, the avian influenza epidemic in my country and neighboring countries in 2004, the global H1N1 influenza epidemic in 2009, and the Ebola epidemic in Africa in 2014 have given society It has had a major impact on stability, economic construction and people's life and health. The frequent occurrence of these new viral infectious diseases has sounded the alarm for us, and it is very important to strengthen its research and monitoring, as well as early prevention and control.

Marburg hemorrhagic fever is a highly fatal infectious disease caused by Marburg virus. Marburg virus and Ebola virus together form the filovirus genus, but belong to two different species. The earliest documented outbreak of Marburg haemorrhagic fever was in Marburg, Germany in 1967. At that time, 31 people were reported to have contracted the virus and 7 of them died. Twenty-five of the 31 infected people became ill from contact with monkeys infected with Marburg virus. After that, it often occurred in some African countries. So far, 12 outbreaks have occurred. The most serious outbreak occurred in Angola from 2004 to 2005. A total of 374 cases were reported, including 329 deaths. MARV is mainly transmitted through close contact, with a case fatality rate of 23% to 90%, and there is no commercial vaccine yet.

The full length of the Marburg virus genome is about 19kb, which is a single-stranded negative-sense RNA virus. The genome encodes seven viral proteins, including nucleoprotein (NP), matrix proteins VP24 and VP40, structural proteins VP30 and VP35, RNA-dependent RNA polymerase (L protein), and glycoprotein 7 (gp7).

At present, the detection methods of Marburg virus mainly include virus isolation, culture and identification, serum neutralization test, enzyme-linked immunosorbent assay (ELISA), nucleic acid detection, etc. For viral diseases, virus isolation and culture identification are the most reliable detection methods. However, the above-mentioned virus isolation needs to be carried out under the conditions of a biosafety level 4 (P4) laboratory, and it takes a long time, and the requirements for the laboratory and experimenters are extremely high, and it is not suitable for wide application and rapid screening at border ports. Serum neutralization test is the main method for virus serological detection, but it also needs to be carried out in P4 laboratories. At the same time, in the detection of the epidemic in 2005, it was found that ELISA, IgM and other serological or immunological detection methods had low sensitivity and high false negative rate.

The sensitivity of the acid detection method has been unanimously recognized, but the conventional PCR operation is complicated, the equipment is huge, and the technical difficulty is relatively high, which is not conducive to on-site diagnosis at ports and on-site detection in epidemic areas. Therefore, it is urgent to develop a new rapid and simple nucleic acid detection method. The patent of the present invention detects Marburg virus by studying a reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) method.

The loop-mediated isothermal amplification method is a new type of constant temperature nucleic acid amplification method, which is characterized in that 4 or 6 primers are designed for six segments on the target nucleic acid, and then reacted at a certain temperature by using a strand displacement reaction. The 3' end and 5' end of the 2 internal primers among the 4 primers can respectively recognize two different regions in the target nucleic acid sequence, and the 5' end sequence can be synthesized by the extension reaction starting from the 3' end Annealing of complementary strand regions. The inner primer can form a stem-loop structure and undergo a self-extension reaction, and then a new inner primer anneals at the position of the loop structure to perform a strand displacement synthesis reaction. The two are continuously repeated in a cycle to realize the amplification reaction. Therefore, even with only one enzyme, the loop-mediated isothermal amplification method can achieve isothermal amplification. When RNA is used as a template, cDNA synthesis and nucleic acid amplification can be completed in one step by adding reverse transcriptase in advance (reverse transcription loop mediated isothermal amplification reaction).

The characteristic of the loop-mediated isothermal amplification gene amplification method is that while synthesizing a large amount of nucleic acid, a large amount of by-product pyrophosphate ions are also generated. Calcein contained in the fluorescent objective reagent is initially in a state of fluorescence quenching due to binding with manganese ions. However, with the progress of the loop-mediated isothermal amplification reaction, because the pyrophosphate ion by the reaction by-product deprives the bound manganese ion, calcein becomes free and emits fluorescence. Furthermore, it combines with magnesium ions in the reaction solution to enhance the fluorescence. According to this principle, the occurrence of loop-mediated isothermal amplification can be easily judged by visual detection of fluorescence.

The loop-mediated isothermal amplification (LAMP) method has the advantages of rapidity, high efficiency, strong specificity, and high sensitivity. The LAMP method is fast and does not require high experimental requirements. It only needs a water bath, does not require aseptic operation, and can detect a large number of samples in a short period of time. It is suitable for laboratory serological diagnosis, customs quarantine, and large-scale disease surveys. In addition, the LAMP assay is more sensitive and the results are more reliable.

At present, the molecular biology detection of Marburg virus is mainly through PCR technology. According to the specific gene fragment V35 of Marburg virus, the present invention designs a group of specific primers. To detect the amplified fragment within one hour, only a constant temperature water bath is required for operation, the method is simple, the sensitivity is high, and the time is short. The detection method of the present invention can be applied to the rapid and sensitive detection of Marburg virus, and can be used as a rapid screening method at ports to improve the accuracy and timeliness of detection and greatly reduce the detection cycle.

Contents of the invention

One of the technical problems to be solved by the present invention is to provide a fast and sensitive RT-LAMP detection method for Marburg virus. The detection efficiency of inspection and quarantine personnel can not only reduce the workload, but also solve the problem of positive and missed detection that may exist in traditional detection methods to the greatest extent, so as to prevent the occurrence of epidemics and the introduction of foreign Marburg virus to the greatest extent.

The fast and sensitive Marburg virus RT-LAMP detection method provided by the present invention comprises the following steps:

(1) RNA extraction from whole blood samples;

(2) RT-LAMP amplification:

a. The RT-PCR reaction system includes: 2× reaction buffer (RM), gene-specific primers for the sample to be tested, dd H2O, enzyme solution, RNA extraction solution, and fluorescent visual reagents;

b. Add the PCR reaction solution into the PCR tube, put it into a 63°C constant temperature instrument or a LAMP detector, and the reaction conditions of the constant temperature reaction: 63°C for 60 minutes. ;

(3) Interpretation of the results: the color of the negative control is brown, and the color of the positive sample is green. If a LAMP detector is used, there will be an obvious amplification curve.

The RNA extraction described in step (1) can use blood sample genome RNA extraction kit to extract RNA. Take 100ul whole blood sample, and extract RNA according to the extraction steps in the kit manual;

The PCR reaction system described in step (2) is: 12.5 μl of 2X reaction buffer, 2.5 μl of specific primer mixture, 1.0 μl of enzyme solution, 3.0 μl of dd H2O, 1.0 μl of fluorescent visual reagent, and 5 μl of RNA extraction solution.

The gene-specific primers for the sample to be tested described in step (2) are three pairs of primers designed based on the loop-mediated constant temperature amplification technology with the specific gene VP35 gene of Marburg virus as the target gene: the inner primer FIP/BIP, the outer primer Primers F3/B3 and loop primers LF/LB were used to compare the sequences of VP35 gene in various Marburg viruses, and to select a section with the highest homology to design primers to improve the specificity of Marburg virus detection. The three pairs of specific primers are:

Internal primer-FIP: 5'-GTTCCTCACTGAGTTCAAGGACCCGGTTAAAAATGCAACAACAG-3' (SEQ ID NO 1)

Inner primer-BIP: 5'-CAAGCCAAACCTCTCAGCCAGGATGTGGAATGGAGTGT-3' (SEQ ID NO 2)

Outer primer-F3: 5'-AGCTTGTAGTAAGGGGACTA-3' (SEQ ID NO 3)

Outer primer-B3: 5'-GTAAGCAATTTTTGAAAGGACC-3' (SEQ ID NO 4)

Loop Primer-LF: 5'-ACATCTTGTCGGCTGCAT-3' (SEQ ID NO 5)

Loop Primer-LB:5'-TTACCCATCTACCCGGCAA-3' (SEQ ID NO 6)

The present invention designs three pairs of specific primers for the specific gene of Marburg virus, so that the detection method of the present invention has higher accuracy in detecting Marburg virus gene, and can detect Marburg virus sensitively and specifically. Compared with the conventional PCR method, the detection Sensitivity and specificity were significantly higher than conventional methods. The detection method of the present invention can not only meet the requirements of daily epidemic safety detection, but also meet the detection requirements of clinical samples. Compared with the existing detection methods, the present invention only needs to complete the reaction in a small-scale LAMP amplification instrument or a constant temperature water bath, and the detection results can be detected by visual inspection, which greatly reduces the time and complexity of detection. The equipment is simple and easy to carry, which greatly improves the current detection capability. The total time from sample collection to detection can be completed within 1 hour, which is beneficial to the rapid and accurate detection of Marburg virus.

Description of drawings

Figure 1 is the detection result of Marburg virus RT-LAMP in Example 1.

The left picture shows the signal intensity detected by the detection tube in the right picture in the LAMP amplification instrument, the right picture shows the color change observed by the naked eye, and the numbers on the right picture represent copies/ul.

Fig. 2 is the detection specificity result of the Marburg virus V35 positive sample of the Marburg virus RT-LAMP detection method in Example 1.

The left is the fluorescence intensity of amplification products of different samples, and the right is the sample names of different histograms.

Fig. 3 is the result of the sensitivity experiment of the Marburg virus RT-LAMP detection method in Example 1.

The left is the sample concentration of each bar graph, and the right is the fluorescence intensity of the amplification products of samples with different concentrations.

Fig. 4 is the detection time measurement result of the Marburg virus RT-LAMP detection method in Example 1.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1 Specificity and sensitivity of Marburg virus RT-LAMP detection method

1. Experimental steps

1. In vitro synthesis of Marburg virus and Ebola virus RNA

Using bioinformatics methods to analyze the genome sequence (Ang1379c) of the published Marburg virus Angola strain, the artificially synthesized fragment (about 600 bp) containing the specific detection Marburg virus VP35 was cloned on the PCR cloning vector (TOPO TA cloning kit), and used Plasmid DNA extraction kit to purify the plasmid DNA, sequence to confirm that the insert is correct, and then perform in vitro transcription: First, digest the plasmid with Not I to linearize it to exclude downstream RNA products, then use MAXIscript T7Kit for in vitro transcription to generate target RNA, and then use TurboDNase Perform DNase treatment to remove untranscribed plasmid DNA templates, and finally use ethanol precipitation to purify the RNA product, which is the positive control RNA template, and store it in a -70°C ultra-low temperature refrigerator until use. The same method was used to obtain the RNA fragment of Ebola virus VP35.

2. Collection of blood samples

Take 1.0ml of whole blood sample from the patient for port testing, and centrifuge to obtain serum.

3. Genomic RNA Extraction

Genomic RNA extraction kit from blood samples (spin column type, Qiagen) was used to extract dengue fever RNA, yellow fever RNA, and West Nile virus RNA. Take 100uL serum sample, and extract RNA according to the extraction steps in the kit instruction manual.

4. Determination of RNA Concentration

The concentration of RNA fragments was determined by Nanodrop 2000c, and the copy number of RNA was calculated according to the size of RNA fragments.

5. Amplification of target genes

5.1 Design of primers

Three pairs of primers were designed based on the loop-mediated constant temperature amplification technology, with the VP35 gene unique to Marburg virus as the target gene: the inner primer FIP/BIP, the outer primer F3/B3, and the loop primer LF/LB. To compare the sequences in different Marburg viruses, select the one with the highest homology to design primers to improve the specificity of Marburg virus detection. The three pairs of specific primers are:

Internal primer-FIP: 5'-GTTCCTCACTGAGTTCAAGGACCCGGTTAAAAATGCAACAACAG-3' (SEQ ID NO 1)

Inner primer-BIP: 5'-CAAGCCAAACCTCTCAGCCAGGATGTGGAATGGAGTGT-3' (SEQ ID NO 2)

Outer primer-F3: 5'-AGCTTGTAGTAAGGGGACTA-3' (SEQ ID NO 3)

Outer primer-B3: 5'-GTAAGCAATTTTTGAAAGGACC-3' (SEQ ID NO 4)

Loop Primer-LF: 5'-ACATCTTGTCGGCTGCAT-3' (SEQ ID NO 5)

Loop Primer-LB:5'-TTACCCATCTACCCGGCAA-3' (SEQ ID NO 6)

5.2 Reaction system

Amplify on a LAMP amplification instrument. The amplification system is: 12.5 μl of 2X reaction buffer, 2.5 μl of specific primer mixture, 1.0 μl of enzyme solution, 3.0 μl of dd H ₂ O, 1.0 μl of fluorescent visual reagent, RNA Extraction solution 5μl.

Reaction conditions for constant temperature reaction: 60°C for 60 minutes.

6. Interpretation of results

a, Amplify on the LAMP amplification instrument, judge by the amplification histogram

b, Amplified in a constant temperature instrument, judged by the color change of the amplification system, the negative control is brown, and the positive is green.

7. Detection system specificity

According to the determination of RNA concentration, two Marburg virus V35 nucleic acid fragment concentration samples of 10E+6copies/ul and 1copies/ul were obtained, respectively with two concentrations of Marburg virus VP35 gene RNA, Ebola virus V35 gene fragment, and dengue virus RNA, yellow fever virus RNA, and West Nile virus RNA were used as templates for detection according to step 5.2, and their specificity was observed.

8. System sensitivity test

Measure each dilution according to the method in 5.2, and calculate the detection limit of the detection method.

9. Detection time: Determine the detection time according to the detection curve

2. Results

1. Detection ability of Marburg virus

Experiments were performed with in vitro synthesized Marburg RNA samples and the results are shown in Figure 1. It is found through experiments that samples ranging from 1 copy/ul to 10 ⁹ copies/ul can be detected, and the detection result is consistent with the result of the color change of the reaction solution observed by the naked eye through the machine-detected fluorescence intensity. It fully demonstrates that the method is simple to operate, as long as there is a water bath, the reaction can be carried out and the results can be observed. Compared with the fluorescence intensity collected by the machine, it may not be as accurate as the machine.

2. Sample specificity test:

As shown in Figure 2, two Marburg virus V35 nucleic acid fragments, 10E+6copies/ul and 1copies/ul, can be detected at both high and low concentrations, while other viral RNAs and Ebola artificially synthesized V35 nucleic acid fragments were not detected.

3. Sensitivity experiment

Detect samples with different dilution concentration gradients of 100copies/ul, 10copies/ul, 1copies/ul, 0.1copies/ul, 0.01copies/ul, 0.001copies/ul and 0.0001copies/ul respectively, and conduct method sensitivity experiments, as shown in Figure 3 , can detect samples with a concentration of 1copies/ul, the sample volume is 5ul, and the detection limit is 5copies.

4. Detection Time Determination

When 10, 100copies/ul samples can be detected at 28min, and 1copy/ul samples can be detected at 42min, see Figure 4 for details. In addition, other data show that 10E+6copies/ul samples can even be detected in 16 minutes, which has excellent timeliness.

Claims (1)

1. the primer based on RT-LAMP rapid sensitive detection Marburg virus, which is characterized in that with Marburg virus VP35 gene Segment is target gene, based on loop-mediated isothermal amplification technology three pairs of specific primers of design are as follows:

Inner primer-FIP:

5 '-GTTTCCTCACTGAGTTCAAGGACCCGGTTAAAAATGCAACAACAG-3 ' (SEQ ID NO.1) inner primer- BIP:

5’-CAAGCCAAACCTCTCAGCCAGGATGTGGAATGGAGTGT-3’(SEQ ID NO.2)

Outer primer-F3:

5’-AGCTTGTAGTAAGGGGACTA-3’(SEQ ID NO.3)

Outer primer-B3:

5’-GTAAGCAATTTTTGAAAGGACC-3’(SEQ ID NO.4)

Ring primer-LF:

5’-ACATCTTGTCGGCTGCAT-3’(SEQ ID NO.5)

Ring primer-LB:

5’-TTACCCATCTACCCGGCAA-3’(SEQ ID NO.6)。