RU2761170C1 - METHOD FOR IDENTIDYING THE RNA OF THE SARS-CoV2 VIRUS USING MULTIPLEX ISOTHERMAL LOOP AMPLIFICATION WITH REVERSE TRANSCRIPTION - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology and, in particular, to genetic engineering. Isothermal loop amplification is executed using specifically selected oligonucleotide primers complementary to the site of the genomic RNA of the SARS-CoV2 coronavirus and the genomic RNA of the MS2 bacteriophage, wherein amplification of two DNA fragments complementary to the genomic RNA of the SARS-CoV2 coronavirus and the genomic RNA of the MS2 bacteriophage is executed simultaneously in one reaction mixture. The amplification results are detected both in real time and after amplification using an intercalating fluorescent dye based on the analysis of the melting curves of amplified fragments.

EFFECT: reduction in the duration of the method for identifying the RNA of the SARS-CoV2 virus.

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Description

The proposed invention relates to the field of biotechnology and, in particular, to genetic engineering and can be used to identify the genetic material (RNA) of the SARS-CoV2 coronavirus for diagnostic purposes.

The SARS-CoV2 virus, belonging to the genus Betacoronavirus, is the cause of the COVID-19 coronavirus infection pandemic, which continues as of October 2020. The pandemic has affected all countries of the world, the total number of infected people, according to the WHO report dated October 20, 2020, was more than 40 million people with more than 1.1 million registered deaths (data as of October 18, 2020), of which 1.45 million infected were registered in Russia. As a result, there is an extremely urgent need for diagnostic tests to detect the RNA of the SARS-CoV2 virus.

The most common test systems used are based on real-time PCR combined with reverse transcription (real-time RT-PCR). RT-PCR allows you to get results within 1.5-2 hours from the start of the RNA analysis. At the same time, due to the dramatically increased workload on diagnostic laboratories and the limited number of special devices for conducting RT-PCR (amplifiers), the analysis time has become a factor limiting the total number of tests carried out during the working day. In other words, in order to increase the throughput of laboratories, it seems necessary to develop faster diagnostic methods.

Over the past 30 years, against the background of the rapid development of molecular biology, many different approaches to amplification of nucleic acids, alternative to PCR, have been developed. Many of them involve avoiding the use of cyclic heating-cooling of reaction mixtures and carrying out DNA amplification at a constant temperature - these are methods of isothermal amplification (NASBA, RPA, LAMP, HDA, MDA, RCA, SDA and a number of other approaches [1-3]. reactions at constant temperature eliminates the need for complex and expensive amplifiers, which makes it possible to miniaturize equipment for carrying out amplification reactions and develop devices for testing outside the laboratory at the patient's bedside (point-of-care). isothermal loop amplification (LAMP, loop-mediated isothermal amplification) [4]. LAMP is based on the use of the chain-displacing activity of some DNA polymerases and 2-3 pairs of oligonucleotide primers. oil or probes, turbidimetrically or electrochemically, both in real time and at the end of the reaction. On the basis of LAMP, many tests have been developed to identify infectious agents in humans, as well as agricultural animals and plants, including influenza, Zika viruses, tuberculosis and malaria pathogens [5-8]. The sensitivity and specificity of LAMP are not inferior to PCR; at the same time, LAMP is more resistant to inhibitors and allows obtaining test results 2-3 times faster (30-40 minutes versus 1.5-2 hours).

Due to its advantages over PNR while maintaining sensitivity and specificity, LAMP is a promising method for creating diagnostic tests for detecting SARS-CoV2 virus RNA. During 2020, several similar tests were created, some of which were certified and used in real clinical practice [9-11]. It should be noted that the main focus of researchers has been on developing LAMP-based tests for bedside diagnostics outside of laboratories. In most studies, real-time detection of LAMP results was used to optimize the methodology and check analytical characteristics, while the detection of the final test results was carried out visually, according to the color of the reaction mixture. At the same time, LAMP can be used in real time within diagnostic laboratories, which will save hardware time and increase test throughput.

Multiplexing - amplification of two or more DNA fragments in one reaction mixture. With the help of multiplexing, it becomes possible to simultaneously detect several pathogens and reduces the cost of testing. In addition, multiplexing achieves simultaneous amplification of the internal control of the reaction or the internal control of DNA or RNA extraction from the sample, which makes it possible to monitor the quality of the isolation of the tested nucleic acid samples and the performance of the reagents for reverse transcription and amplification. However, due to the specific nature of the amplification process and the LAMP reaction products, its multiplexing is a difficult task. Thus, DNA polymerases used in LAMP lack 5'-3'-exonuclease activity, which makes it impossible to use hydrolysable fluorescently-labeled probes. The LAMP product is a concatemeric sequence composed of repeated amplified fragments, which limits the length separation of amplification products without additional manipulation. LAMP multiplexing is possible using modified oligonucleotides, including fluorescently labeled ones, or by melting amplification products in the presence of intercalating dyes [12-14]. In the latter case, the differentiation of products occurs due to the characteristic melting temperatures for each product.

The closest to the claimed method - the prototype, is a method for detecting the DNA of S. aureus, L. monocytogenes and S. spp, by isolating DNA from the analyzed sample, conducting a multiplex LAMP with three sets of primers complementary to the regions of the genomes of these organisms in one test tube using LAMP melting at high resolution in the same tube of amplification products. At the last stage of the method, high-resolution melting results are analyzed and the melting temperatures of the amplified fragments are determined. The presence of microorganism DNA in the analyzed sample is established by the appearance of a characteristic melting peak corresponding to the amplification product of the microorganism genome region [12].

The disadvantage of this method is its duration, associated with the time spent on melting with high resolution, which is several times longer than the time spent on carrying out conventional melting of DNA fragments.

The objective of the invention is to develop a faster method for detecting RNA of the SARS-CoV2 virus using multiplex isothermal loop amplification coupled with a reverse transcription reaction.

EFFECT: shortening the duration of the method for detecting the RNA of the SARS CoV2 virus.

The task is achieved by the proposed method, which is as follows.

Isothermal loop amplification (LAMP) of the RNA isolated from the analyzed sample, to which the MS2 phage is preliminarily added, is carried out using specially selected oligonucleotide primers complementary to the region of the genomic RNA of the SARS-CoV2 coronavirus and the genomic RNA of the bacteriophage MS2. Amplification of two DNA fragments complementary to the genomic RNA of the SARS-CoV2 coronavirus and the genomic RNA of the bacteriophage MS2 is carried out simultaneously in the same reaction mixture using reverse transcriptase and a large fragment of Gss polymerase. The amplification results are detected using an intercalating fluorescent dye. Direct amplification is carried out in a thermal cycler for conventional PCR, or in a real-time PCR device. The presence of SARS-CoV2 coronavirus RNA in the analyzed sample is established by analyzing the melting curves of the amplified fragments by the appearance of a melting peak with a specific temperature.

Conserved regions of the genomic RNA of the SARS-CoV2 virus encoding a region of protein E, as well as a region of the genome of the MS2 phage, were selected as targets for the LAMP primers. Primers were selected in accordance with the recommendations posted on the primerexplorer.jp website. The efficiency of amplification using the selected primers was assessed by performing LAMP with the RNA fragments of the SARS-CoV2 virus and the MS2 phage. The concentration of RNA standards was measured by digital drop PCR on a QX200 platform (Bio-Rad; USA).

The developed new method for detecting SARS-CoV2 coronavirus RNA allows real-time detection of the genetic material of the virus within 40 minutes with a detection limit of at least 20 copies of viral RNA in the reaction mixture, together with the simultaneous amplification of the MS2 control RNA fragment.

The invention is illustrated by the following specific examples.

Example 1.

LAMP was carried out in a reaction volume of 20 μL containing 1 × reaction buffer for Bst polymerase (20 mM Tris-HCl pH 8.8, 10 mM (NH ₄ ) ₂ SO ₄ , 150 mM KCl, 0.1% Tween-20, 2 mM MgSO ₄ ), 1.4 mM each dNTP, 1x primers for SARS-CoV2 (0.2 μM external primers (F3 / B3) each, 0.6 μM loop primers (LF / BF), 1.6 μM internal primers (FIP / BIP)), 0.5x primers on MS2 (0.1 μM external primers (F3 / B3) each, 0.3 μM loop primers (LF / BF), 0.8 μM internal primers (FIP / BIP)), the sequences of which are presented in Table 1, RNA template (the type and amount of the template are indicated below), 100 e.a. reverse transcriptase M-MuLV, 2 ea. a large fragment of Gss polymerase from Geobacillus sp. 777 [15], intercalating dye SYTO-82 to a concentration of 1 µM. The reaction was carried out in a CFX96 thermocycler (Bio-Rad; USA). The program included the following stages: 10 min of reverse transcription at 50 ° С, annealing of primers and elongation at a temperature of 64 ° С for 90 cycles of 20 s each with registration of a fluorescence signal on the FAM channel; determination of the melting temperature of amplification products in the range of 70-95 ° C after amplification to determine the accumulated amplification products. The results of isothermal amplification were assessed by the Tt parameter (time-to-threshold - the time to the intersection of the accumulation curve of the amplification product with the threshold value) and the melting curves of the LAMP products.

The detection limit for SARS-CoV2 coronavirus RNA using multiplex LAMP was estimated by varying the amount of CoV2-E RNA within 100-10 copies against the background of 12000 copies of MS2 RNA in the reaction mixture.

A total of 4 concentrations of CoV2-E RNA were used: 100, 50, 20, and 10 copies in the reaction mixture; with each concentration, LAMP was carried out with primers MS2-CoV2-E in 20 technical repetitions in one run. The presence of CoV2-E RNA was determined by the appearance of a corresponding peak on the melting curve of the amplification products. The results of the analysis of the melting curves of the LAMP products when setting the detection limit of the MS2-CoV2 duplex are shown in FIG. 1. In FIG. 1 shows the characteristic melting peaks products LAMP, obtained monopleksom CoV2-E and March ¹⁰ RNA copies CoV2-E (highlighted squares), with monopleksom MS2 and March ¹⁰ RNA copies MS2 (highlighted rhombuses), the duplex MS2-CoV2-E 12000 copies to the reaction of MS RNA and CoV2-E RNA: 100 copies (marked with circles), 50 copies (marked with triangles) and 20 copies (marked with crosses).

The CoV2-E melting peak was present in all 20 technical replicates for CoV2-E RNA concentrations of 100, 50, 20 molecules in the reaction mixture, as well as in 16 out of 20 technical replicates for CoV2-E RNA concentrations of 10 molecules per reaction. Thus, the detection limit of the multiplex LAMP was no less than 20 SARS-CoV2 RNA molecules in the reaction mixture. The obtained detection limit corresponds to those for previously published test systems for detecting RNA SARS-CoV2 coronavirus.

Example 2.

Validation of the proposed method (multiplex LAMP MS2-CoV2-E) was carried out on 40 clinical samples of nasopharyngeal swabs obtained from patients of CNMT ICFBM SB RAS. All participating patients signed informed consent to conduct the study. RNA was isolated from the samples using a set of reagents for the isolation of RNA / DNA from clinical material "RIBO-prep", after which testing was carried out by two methods: RT-PCR in real time using primers recommended by WHO [16] (Table 1) and multiplex LAMP MS2-CoV2-E. The results of a comparison of multiplex LAMP MS2-CoV2-E with real-time RT-PCR are presented in Table 2.

Out of 40 samples, the results of testing by both methods coincided for 37. Moreover, for two negative by RT-PCR samples tested as positive by LAMP, the Cq of RT-PCR in real time exceeded 35. Thus, the concordance of the results of multiplex LAMP with RT- Real-time PCR was 92%.

Thus, the developed new method for detecting SARS-CoV2 coronavirus RNA makes it possible to detect in real time the genetic material of the virus within 40 minutes with a detection limit of at least 20 copies of viral RNA in the reaction mixture, together with the simultaneous amplification of the control MS2 RNA fragment.

Sources of information:

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Claims (2)

1. A method for detecting RNA of the SARS-CoV2 virus using multiplex isothermal loop amplification with reverse transcription, including the isolation of RNA from the analyzed sample, carrying out isothermal loop amplification using oligonucleotide primers, followed by detection of the amplification results by melting the amplification products, characterized in that the amplification is carried out in that two DNA fragments complementary to the genomic RNA of the SARS-CoV2 coronavirus and the genomic RNA of the bacteriophage MS2, while amplification is carried out simultaneously in one reaction mixture using reverse transcriptase, a large fragment of Gss polymerase, a set of oligonucleotide primers presented in Table 1, and an intercalating dye, and the detection of SARS-CoV2 coronavirus RNA is carried out by analyzing the melting curves of the amplified fragments by the appearance of a melting peak with a specific temperature. 2. The method according to claim 1, characterized in that the genomic RNA of the bacteriophage MS2 is added to the analyzed sample before amplification.

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