WO2014081323A2

WO2014081323A2 - Method of rna viruses identification and its application

Info

Publication number: WO2014081323A2
Application number: PCT/PL2013/050032
Authority: WO
Inventors: Krzysztof PYRĆ; Karol STOŻEK; Jan Potempa
Original assignee: Uniwersytet Jagielloński
Priority date: 2012-11-22
Filing date: 2013-11-22
Publication date: 2014-05-30
Also published as: PL401707A1; PL225633B1; WO2014081323A3

Abstract

The invention relates to a method for the identification of RNA viruses involving the use of short, conservative 4-10 nucleotide RNA stretches as primer attachment sites in a reverse transcription reaction. During this process synthetic adapters are added to the 3' end of the amplicon. The method also includes the synthesis of a second DNA strand, which allows for the addition to the nucleic acid fragment of the synthetic adapters on the 5' end of amplicons. Both 5' and 3' flanking adapters are subsequently used for amplification of the resulting genetic material. The invention also relates to the application of a method of the identification of RNA viruses, including new pathogens, but also known pathogens, which due to their natural variability were not detected with other methods.

Description

Method of RNA viruses identification and its application

The invention relates to a new method for the identification of RNA viruses involving the use of short 6-8 nucleotide RNA elements in the viral genome, as the attachment sites for specific primers in a reverse transcription reaction. Method also includes the synthesis of the second DNA strand, which allows for the addition of the synthetic adapters to the nucleic acid fragment, enabling for subsequent amplification. This is a modification of the currently applicable standard PCR reaction.

In particular the invention includes the use of method for the identification of RNA viruses, including known pathogens and new pathogens, which, due to the natural variability may not be detected by the standard methods.

Viral infections are associated with numerous human and animal diseases. However, due to the difficulties in the identification of viruses and their high variability, the etiological factors of numerous diseases remain unknown. The perfect example are respiratory diseases, as the continuous research still leads to the identification of new pathogens infecting the respiratory tract. This includes pathogens that were circulating in human population for years (e.g., human coronavirus NL63 or HKU1 , human bocavirus), but also pathogens that recently crossed the species border and pose a threat to human population (e.g., severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV)). The problem with their identification results directly from high variability of viruses and an imperfect detection system.

A method known in the art and normally used to amplify the genetic material is the polymerase chain reaction (PCR), which was developed by Mullis et al. and described (US4683195). There are also methods to identify the unknown viruses. The oldest known method is an electron microscopy, used to visualize a virus in infectious material. However, this method does not allow for the molecular characterization of pathogen, and therefore it is commonly used as a supplementary technique. Currently used methods for the identification of known viruses include a variety of techniques, from the cell culture and testing for the presence of viral antigens, to molecular methods allowing for the amplification of nucleic acids.

Molecular methods also include techniques to identify the new viruses or variants of viruses which are undetectable by other means. These encompass most promising methods for the amplification of the genetic material regardless of its sequence and methods that rely on the presence of the nucleic acid sequences which are present in a similar location in the genomes of all pathogens belonging to a particular group / family. The first group includes methods such as a random amplification, differential display or viruses identification methods based on the common presence of restriction sites (e.g., VIDISCA or SISPA methods). In the abovementioned techniques, an amplification of nucleic acids is not dependent on the presence of specific primer attachment site. For example, primers used for the random amplification contain a random sequence, which is able to hybridize with virtually any nucleic acid template, including viral nucleic acids. The differential display is more specific, and uses arbitrarily chosen primers (fixed sequence) to amplify the random nucleic acid fragments (W09813521 , US20030725969, Welsh 1990). Optimization (choosing the right pair of primers), allows also for the amplification of viral nucleic acids. Comparison of the resulting amplicons in a tested and a reference sample leads to the selection of DNA fragments that may be derived from pathogens. Another methodology is used in VIDISCA and SISPA methods (WO9808981 , US2005175997, US2004259109, US2003175908, van der Hoek 2004, Reyes 1991). In this case, an amplification of nucleic acids is based on the common presence of restriction sites. A detailed analysis of the frequency of occurrence of restriction enzymes (according to the cutting frequency) allows to select sites that any sufficiently long nucleic acid molecule will contain. Following enzymatic conversion of RNA and a single- stranded DNA to a double-stranded DNA, the nucleic acids are treated with the selected restrictase. The resulting cleavage sites are then used to ligate synthetic DNA adapters of known sequence. In the next steps these adapters are used for the specific amplification of the full range of nucleic acids. Again, a comparison of the resulting amplicons in the tested and a reference sample allows selecting DNA fragments present exclusively in the tested (infectious) material. Unfortunately, all the above described methods are labor-consuming and time-consuming, and they require highly skilled personnel. These methods are difficult to use in a standard diagnostic laboratory.

The most sensitive method for the identification of viruses is the use of universal primers in a standard PCR amplification of nucleic acids. This methodology makes use of the presence of conserved sites in the genomes of viruses belonging e.g., to a single family. The use of such sites for primer design allows to hope that in the case of unknown viruses, these primer attachment sites will also be present and it will be possible to amplify and identify new viruses and new variants of known viruses. The biggest disadvantage of this method is that it requires primer attachment sites of 18-25 nucleotides. Due to high variability of viral species in some cases it may not be possible. A certain convenience in this case is the CODEHOP method, which allows the use of conservative protein sequence for the design of highly degenerated DNA primers (Rose 1998).

The aim of the method according to the invention is to provide a new method for the identification of RNA viruses, and its application. The developed method is much more specific compared to methods based on random amplification and thus it is much easier to implement. Furthermore, it shows a broader specificity in comparison with standard PCR using the universal primers. In the developed method a modification compared to a standard PCR is the addition of synthetic DNA adapters at the stage of reverse transcription and second strand DNA synthesis, which allow for subsequent amplification of nucleic acids.

Detailed description of the invention

The essence of the invention is a method for the identification of RNA viruses characterized in that it comprises the following steps:

(a) purified RNA is transcribed into cDNA in a reverse transcription reaction, involving a primer consisting of DNA fragment having a sequence that may anneal to a conservative region of viral sequences with a length of 4-10 nucleotides and a DNA fragment forming the adapter of known sequence, which is then used as primer annealing site in a PCR reaction;

(b) obtained as a result of step (a) RNA-DNA duplex is enzymatically digested in order to obtain a single-stranded DNA;

(c) obtained in step (b) a single-stranded cDNA is enzymatically transcribed into a double-stranded DNA using an enzyme allowing the primer-initiated DNA synthesis on a DNA template using a primer for the synthesis of the second strand (SS) with a similar structure as the primer for a first-strand DNA synthesis (RT) with a sequence that may anneal to a conservative region of the viral sequences with 4-10 nucleotides in length, and the adapter with a known sequence used for DNA amplification in the subsequent PCR reaction is to the 5 'side;

(d) obtained in step (c) a double-stranded DNA from both sides is flanked with DNA fragments of known sequences, which are used to amplify the DNA in a first PCR reaction with primers having the same sequence as the RT and SS primers, or a sequence homologous to any RT and SS primer fragment;

(e) obtained in step (d) product is used in a second PCR reaction using primers with a sequence homologous to the sequences of the RT and SS primers and containing at least one nucleotide more from the 3 ' side in comparison to the RT and SS primers;

(f) obtained in step (e) product is subjected to an analysis which allows to assess the size of the resulting DNA fragment.

Preferably, when the sequence that may anneal to a conservative region of viral sequences in step (a) and step (c) has a length of 4-10 nucleotides.

Also preferably, when in the implementation of the method according to the invention, the replication of the nucleic acid takes place at temperature below 50°C, allowing the efficient and specific binding of the primers to the complementary short sequences in the viral genome, or its equivalent in the form of single-stranded DNA.

Preferably, when the RNA-DNA duplex is digested enzymatically with RNase H. Also preferably, when the distance between the RT and SS primers is in the range of 50-300 nucleotides.

Preferably, when the nucleic acid replication is carried out in a reverse transcription and a second strand DNA synthesis reactions.

Also preferably, when the product obtained in step (e) is subjected to an analysis which allows to assess the size of the DNA fragment obtained as a agarose gel electrophoresis or polyacrylamide gel electrophoresis or sequencing. This type of analysis can be carried out e.g., using agarose or polyacrylamide gel electrophoresis or any other method that allows determination of the DNA length and/or nucleotide sequence (e.g., sequencing).

The subject of the invention is also the use of the presented method for the detection of known RNA viruses, variants of known RNA viruses, which may not be detected by standard methods, as well as to detect new RNA viruses. In addition, the method according to the invention can be used to amplify any RNA fragment containing in its sequence at least two 4-10 nucleotide long conservative regions, which may serve as primer annealing sites.

The method according to the invention is similar to the amplification method using the universal primers due to the need to determine the presence of regions conservative for all viruses that are to be detected by an assay. The main difference lies in the fact that these regions are shorter in the case of the described method than in the universal primer method and they allow the identification of the virus with the use of conservative sites of 4-10 nucleotides in length.

A brief description of the drawings

The subject of the invention is described below in detail by referring to the attached drawing, in which:

Fig. 1 illustrates the principle of the method according to the invention, in which the reverse transcription and the second strand DNA synthesis are carried out on starting material (total RNA: black color) in the presence of a primer consisting of short elements that may anneal to conserved sites within a given group of viruses (respectively, dark green and dark blue for the RT and SS primers) and longer synthetic adapter which may serve as a primer attachment site during subsequent PCR amplification (respectively, light green and light blue for the RT and SS primers).

Fig. 2 illustrates the example of 5' primers for use in reaction of RNA amplification according to the described method. Blue color indicates the region in primers that may be used as primer attachment site during subsequent PCR amplification. The red color indicates the fragment homologous to the viral genome in highly conservative region (an example based on the genome of HCoV-NL63); note: RT/SS and first PCR primers may be identical. Fig. 3 illustrates the action of the method according to the invention in the form of virus detection (HCoV-NL63 and HCoV-HKUI ), where M is a size marker, W is water, and NL63 and HKU1 : Control (-) and infectious (+)samples prepared from the cell culture material.

Fig. 4 illustrates the sensitivity of the method according to the invention, wherein the dilution of the virus was analyzed containing 10⁹, 10⁸, 10⁷, 10⁶ and 10⁵ copies of viral RNA/ml; for each sample three different DNA polymerases on a DNA template were used, namely: DNA polymerase I (A), T7 DNA polymerase (B) and sequenase (C).

Fig. 5 illustrates the specificity of the method according to the invention; analysis was performed on samples containing various human RNA and DNA viruses, including human metapneumovirus (hMPV), human adenovirus (ADV), rhinovirus (RV), enterovirus (EV), influenza virus type A and B (respectively IAV and IBV), parainfluenza virus types 1 , 2 and 3 (respectively P1 , P2 and P3), and the respiratory syncytial virus (RSV); M denotes the DNA size marker.

Fig. 6 illustrates the detection of HCoV-NL63 in different clinical samples, where samples of sputum, bronchoalveolar lavage fluid and nasal washes have been spiked with viable HCoV-NL63 (TCID₅₀ of 400); there was no significant inhibition of the reaction in any of the materials as well as there was no appearance of additional bands; W stands for water, while characters "-" and "+" respectively denote samples not containing or containing human coronavirus NL63.

Examples of the implementation of the invention

Example 1. Detailed description of the implemented method according to the invention

In a first reaction step, the purified RNA is transcribed into cDNA in a reverse transcription reaction. In this case, the RT primer is used (a common primer for a reverse transcription reaction and the first PCR reaction). This primer consists of a short sequence that may anneal to a conservative region in the viral genome and a longer adapter fragment with a known sequence used for subsequent PCR amplification (Fig. 1). A DNA primer consisting of the 4-10 nt long fragment with sequence that may anneal to a conservative region of the viral sequence and a longer (16-20 nt) adapter with known sequence, which is used as primer attachment site in a subsequent PCR reaction.

After the reverse transcription reaction RNA-DNA duplex is digested enzymatically. In this case it is preferred to use an enzyme such as RNase H, which specifically digests the RNA strand, without disrupting DNA strand. Then, the resulting single-stranded cDNA is enzymatically transcribed into a double-stranded DNA using an enzyme allowing the primer- initiated synthesis of DNA on the DNA template. In this reaction, a primer is used for the synthesis of the second strand (SS), which is designed similarly as the primer for the synthesis of first-strand DNA (RT). This primer consists of a short (4-10 nt) region with a sequence that may anneal to the conservative region in the viral genome. This conserved site should be located upstream (closer to the 5' terminus) in the genome (in the original template) to the RT primer attachment site. The SS primer also contains the adapter region (16-20nt) with a known sequence. This adapter region is used in the subsequent PCR amplification as primer attachment site (Fig. 1). In this case, RT and SS primers should be designed in such a way that the distance between them is less than 300 nucleotides and more than 50 nucleotides.

The resulting double-stranded DNA from both sides contains synthetic DNA fragments of known sequence (adapter regions), that are used as primer attachment sites in a first PCR reaction. During amplification described above primers RT and SS are used (or other primers able to anneal in the adapter regions). Product of first PCR amplification is used in a second PCR reaction (nested PCR). In this process primers able to anneal with the adapter sequence are used, though these are extended on their 3' ends, protruding from the original conserved sequence into the viral genome. The amplification in the second stage allows to increase the specificity of the reaction and generation of amplicons, which can be subjected to gel or sequence analysis (Fig. 1 and Fig.2).

Example 2. Acitivity of the method for coronaviruses

RNA was isolated from an infectious sample containing viral particles of human coronavirus NL63 (cell culture of the monkey kidney; LLC-MK2 line, ATCC number: CCL-7) and human coronavirus HKU1 (culture on fully differentiated primary human respiratory epithelial cells) using commercially available total RNA mini kit (A&A Biotechnology) for isolation of viral RNA. Purified RNA was suspended in 100 μΙ of nuclease-free water (Sigma- Aldrich). After isolation, samples were incubated with DNase (DNase Turbo, Thermo Scientific) for 30 minutes which is sufficient for the complete removal of contaminating DNA. In the example a method for increasing RNA concentration in the sample was used. After isolation, RNA was precipitated using three volumes (300μΙ) of the isopropanol in the presence of 30μg glycogen (Life Technologies). The precipitation reaction was carried out at 20°C for 16 hours. After precipitation, samples were centrifuged (12000 ^χ g, 45 min.) and resulting pellets were washed with 70% ethanol and dried. The resulting RNA was dissolved in 5 μΙ of nuclease-free water (Sigma-Aldrich) and used as a starting material for the reaction.

The whole RNA obtained in the isolation was mixed with 1.5 picomolar (pM) RT primer (5'-CCA AGG GAT TCC CCT YCC CAA AAC -3 '), incubated in a total volume of 6.5 μΙ at 65°C for 5 minutes, cooled with ice and mixed with 3.5 μΙ of solution containing 25 units of MultiScribe reverse transcriptase (Life Technologies), 1 ^χ concentrated buffer for the Polymerase I (Thermo Scientific), 0.4 μΙ 100 millimolar (mM) deoxyribonucleotides (dNTPs) and 0.2 μΙ of dimethyl sulfoxide. The reverse transcription reaction was carried out for 120 minutes at 37°C, and then the enzymes were inactivated by heating up to 85°C for 5 minutes.

The resulting a single-stranded DNA (10 μΙ) was used as a template for synthesis of a second strand DNA. The second strand DNA synthesis reaction was carried on in the same tube, without a purification step. First, the cDNA sample was subjected to thermal denaturation (95°C for 1 min.) and cooled on ice. Sample was subsequently mixed with reaction mixture to reach the total volume of 5 μΙ. The sample was composed of 0.5 units of RNase H, 0.5 μΙ of 10 ^χ concentrated buffer polymerase I (Thermo Scientific), 4.5 units of polymerase I (Thermo Scientific), 0.1 μΙ of dimethylsulfoxide and 3pm SS primer (5'-GCA AGA AAT TCC GAA CTA TGA TSA -3 '). The samples were incubated for 120 minutes at 15° C, and then a double-stranded DNA was isolated from the mixture using a mixture of phenol: chloroform: isoamyl alcohol (pH 8.0). The resulting DNA was precipitated using 300 μΙ of isopropanol (16 h, -20°C), centrifuged (12,000 ^χ g, 45 min.), washed with 70% ethanol, dried at room temperature and suspended in 5 μΙ of nuclease-free water (Sigma-Aldrich).

The resulting a double-stranded DNA was used directly for the 1 ^st PCR amplification using aforementioned RT and SS primers. Reaction was carried out with DreamTaq PCR Master Mix (Thermo Scientific) in a total volume of 20μΙ. Reaction mixture contained 1 pM of RT primer, 1 pM of SS primer and 5 μΙ of double-stranded DNA. The thermal profile of performed reaction is shown in Table 1 .

Table 1

A second PCR reaction was carried out after a first PCR reaction. 5 μΙ of the first PCR product was used in a second PCR reaction in the presence of 5'-PCR2 (5 -AAG AGA TCT ATC CAA ATG ATT ATS -3 ') and 3' PCR2 (5'-CCA TTC AAG GGA CAA ACC TYC CA -3 ') primers. The reaction was performed in a total volume of 20 μΙ using DreamTaq PCR Master Mix (Thermo Scientific) kit. Reaction mixture contained 12 pM of 5'PCR2 I primer and 12pM of 3'PCR2 primer. The thermal profile of reaction is shown in Table 2. Table 2

* In each cycle, the temperature was lowered by 1°C.

After the reaction, the whole mixture was loaded onto a 1.5% agarose gel and the resulting DNA fragments were visualized using a ethidium bromide. The results are shown in Figure 3. The amplification led to formation of DNA fragment of the correct size (about 169 nucleotides) for both human coronavirus NL63 and human coronavirus HKU1 . No signal was detected in control samples (samples from uninfected cell cultures). The sequencing of the resulting DNA confirmed its identity and showed that specific amplification of viral RNA using this method is possible.

Example 3. Sensitivity of the method

To assess the sensitivity of the method, the concentration of RNA in the samples containing starting HCoV-NL63 (cell culture) was assessed using a real-time quantitative PCR (qPCR). Reaction was performed using reverse transcribed RNA (cDNA). cDNA was used in the reaction with High Capacity cDNA Reverse Transcription Kit (Life Technologies), 900 nM primers listed in Table 3 and FAM (6-carboxyfluorescein) / TAMRA (6-carboxy- tetramethylrhodamine) labeled probe (200 nM). Assessment was carried on as previously described (Milewska 2012).

Table 3

Name of primer /

probe Sequence of primer / probe (5'-3')

Primer 5' [63NF2] AAA CCT CGT TGG AAG CGT GT

Primer 3' [63NR1] CTG TGG AAA ACC TTT GGC ATC

FAM-ATG TTA TTC AGT GCT TTG GTC CTC GTG AT-

Probe [63NP]

TAMRA The reaction was carried out using qPCR apparatus (Applied Biosystems, 7500fast) with the following thermal profile (Table 4).

Table 4

After determination of viral RNA concentration in the test samples, dilutions of virus suspension were prepared with assumed concentration of viral genomes ranging from 10⁹ to 10⁵ copies of viral RNA per ml. These samples were used in the analysis. Reaction was carried out as described in Example 2, except that there three different DNA polymerases were used: DNA polymerase I (A), T7 DNA polymerase (B) and sequenase (C). The results are presented in Figure 4, and show that the use of polymerase I (A) and T7 polymerase (B) allowed to obtain a higher sensitivity than the use of Sequenase (C).

Example 4. Specificity of the method

In order to assess the specificity of the method according to the invention, a set of primers described in Example 2 was used. As an input material nucleic acids isolated from infectious samples containing various DNA and RNA viruses. The analysis was carried out as described in Example 1 and Example 2, using RNA isolated from cell cultures infected with human metapneumovirus (hMPV), adenovirus (Adv), rhinovirus (RV), influenza viruses A and B (IAV and IBV), parainfluenza viruses 1 , 2, 3 (respectively P1 , P2 and P3) and RS virus (RSV), the control cells (Con), and water (W). According to Fig. 5, there was no cross- reactivity, i.e., no products of the same size were detected in samples lacking coronaviral nucleic acids. Amplification of virus HCoV-NL63 and HCoV-HKUI resulted in generation of a product of the desired size. This demonstrates that the developed method is highly specific.

Example 5. Activity of the method in clinical specimens

The described method is designed to identify the viral RNA in clinical samples. Therefore, an analysis was performed on various clinical samples negative for coronaviruses, including sputum (P), bronchoalveolar lavage fluid (BALF) and nasal washes (N), infected with infectious material derived from the cell culture (TCID₅₀ of 400, which roughly corresponds to MOI of 0.005). The analysis was carried out as described in Example 1 and Example 2. The amplification products were analyzed on an agarose gel, whose picture is shown in Figure 6. No inhibition of the reaction was observed in clinical samples. References

1. Milewska, Ciejka J et al. (2012) Antiviral research, DOI 10.1016/j. antiviral.2012.1 1.006).

2. Reyes GR, Kim JP (1991 ). Mol Cell Probes, 5(6): 473-81 .

3. Rose TM, Schultz ER , Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S. (1998).

Nucleic Acids Res, 26(7): 1628-35.

4. van der Hoek L, Pyre K ef al. (2004). Nat Med, 10(4): 368-73.

5. Welsh J, McClelland M. (1990). Nucleic Acids Res, 18(24): 7213-8.

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Claims

1. A method for the identification of RNA viruses characterized in that it comprises the following steps:

a. purified RNA is transcribed into cDNA in a reverse transcription reaction, using a primer containing on its 5' end a 4-10 nucleotide long DNA fragment that may anneal to a conserved region present in all pathogens that belong to a certain group and a on its 3' end a DNA fragment of known sequence that may serve as primer attachment site in subsequent PCR reaction;

b. obtained in step (a) RNA-DNA duplex is enzymatically digested in order to obtain a single-stranded cDNA;

c. obtained in step (b) a single-stranded cDNA is enzymatically transcribed into a double- stranded DNA using an enzyme allowing the primer-initiated DNA synthesis on a DNA template using a second strand primer (SS). The SS primer is designed similarly as the primer for a first-strand DNA synthesis (RT). SS primer contains on its 5' end a 4-10 nucleotide long DNA fragment that may anneal to a conserved region present in all pathogens that belong to a certain group and a on its 3' end a DNA fragment of known sequence that may serve as primer attachment site in subsequent PCR reaction;

d. double-stranded DNA obtained in step (c) is flanked from both sides with synthetic DNA fragments of known sequences. This regions are used as primer attachment sites in a first PCR reaction. Primers RT and SS are used in the 1^st PCR reaction; alternatively, primers homologous to any RT and SS primers fragment may be used in the reaction; e. product obtained in step (d) is used in a second PCR reaction using primers with a sequence homologous to the sequences of the RT and SS primers and containing at least one nucleotide more from the 3 ' side in comparison to the RT and SS primers; f. product obtained in step (e) is subjected to analysis which allows to assess the size and/or sequence of the resulting DNA fragment.

2. A method according to claim. 1 , characterized in that a sequence that may anneal to a conservative region of the viral sequence in step (a) and step (c) has 4-10 nucleotides in length.

3. A method according to claim. 1 , characterized in that the replication of the nucleic acid takes place at temperature below 50°C, allowing the efficient and specific binding of the primers to short complementary sequences in the viral genome, or its equivalent as a single-stranded DNA. A method according to claim. 1 , characterized in that the RNA-DNA duplex is enzymatically digested with RNase H.

A method according to claim. 1 , characterized in that the distance between the RT and SS primers is in the range of 50-300 nucleotides.

A method according to claim. 1 , characterized in that the replication of a nucleic acid is a reverse transcription and a second strand DNA synthesis.

A method according to claim. 1 , characterized in that the product obtained in step (e) is subjected to an analysis which allows assessment of the size of the resulting DNA fragment.

Application of method according to claim from 1 to 7 for the detection of known RNA viruses, variants of known RNA viruses, which may not be detected by standard methods, as well as to detect new RNA viruses.

Application of method according to claim from 1 to 7 for the amplification of any RNA fragment containing in its sequence at least two conservative region of 4-10 nucleotides in length for the primer annealing with the appropriate structure.