KR101815105B1 - Hantavirus Whole Genome Sequence Identification Methods Using a Multiplex PCR-based Next-Generation Sequencing and Uses Thereof - Google Patents

Hantavirus Whole Genome Sequence Identification Methods Using a Multiplex PCR-based Next-Generation Sequencing and Uses Thereof Download PDF

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KR101815105B1
KR101815105B1 KR1020150144875A KR20150144875A KR101815105B1 KR 101815105 B1 KR101815105 B1 KR 101815105B1 KR 1020150144875 A KR1020150144875 A KR 1020150144875A KR 20150144875 A KR20150144875 A KR 20150144875A KR 101815105 B1 KR101815105 B1 KR 101815105B1
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이대상
송동현
정성태
송진원
김원근
명현군
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국방과학연구소
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Abstract

The present invention relates to a method for obtaining a hapta virus full-length genome sequence using multiplex PCR-based next generation sequencing and its use. The methods, kits, and compositions of the present invention utilize multiplex primer set pools comprised of sets of multiplex primers comprising forward and reverse primer pairs capable of amplifying some genomic sequences of the desired virus, It can be checked quickly and easily whether or not it exists. In addition, two fragments adjacent to each other in the 5'-to-3 'direction in the 100 to 200 bp long sequence fragments obtained by the primer pairs included in the multiplex primer set include an overlapping sequence . Accordingly, when the fragments amplified by the multiplex primer set pool are all collected and overlapped / linked in the 5'-to-3 'direction, the full-length genome sequence of the target virus can be easily obtained. Thus, the methods, kits and compositions of the present invention enable highly accurate and rapid acquisition of the full-length genomic sequence of a target virus (e.g., Hanta virus) with high-depth coverage, Can be more effectively applied to the diagnosis of cancer.

Description

[0001] The present invention relates to a method for obtaining a hapta virus full-length genome sequence using multiplex PCR-based next generation sequencing and a use thereof. [0002]

The present invention relates to a method for diagnosing the presence or absence of a pathogenic HPV infection from a sample suspected of having hemorrhagic fever (epidemic hemorrhagic fever) using Next-Generation Sequencing (NGS) . More specifically, in the sample pretreatment process for determining the genome sequence of hantaviruses, Hantavirus-specific multiplex multiplex primer gene amplification method was used to increase the initial concentration of the sample, and NGS was used to amplify the whole genome sequence of hantavirus And to a technique for securing it.

The techniques for determining the sequence of the nucleotide sequences of living organisms are largely divided into the first-generation Sanger method and NGS. The first-generation method is also called Sanger sequencing, which was developed in about 1977. In the course of polymerase chain reaction (PCR), de-deoxynucleotide triphosphate (ddNTPs) It is a method to determine the order of nucleotide sequence by collecting amplified DNA fragments by applying the chain-terminating principle in which the synthesis of DNA strands is interrupted. This method has high accuracy, but it takes a lot of time to determine the sequence, is slow, and expensive.

NGS technology was developed by Ilumina and Roche in 2006 and 2007, respectively, as low-cost, high-speed and high-capacity nucleotide sequencing technologies were required due to the impact of the Human Genome Project completed in the early 2000s. NGS technology has started to get popular in the market. It has the advantage of using micro or nanotechnology to reduce the length of the sample and to analyze millions of sample fragments in parallel. The basic principle of NGS technology is to amplify the DNA sequence and to identify the order of the nucleotide sequence through the process of imaging the fluorescence with the camera during the sequence determination process. According to the PCR amplification method, IL-12 uses solid-phase amplification, while Roche 454 and Life Technologies use emulsion PCR (emPCR) .

There are also various company platforms in NGS technology. The NGS equipment used in the present invention is MiSeq developed by Illuminator. MiSeq is a miniature NGS device. It features not only the amplification of the DNA sequence of HiSeq, which is a conventional large sequencer, but also the solution from other companies (Roche, AB) It forms a sequence group. The group of clusters thus formed is sequenced by group, converted to base sequence information of each read, and passed on to the analysis process. MiSeq equipment has succeeded in miniaturization while maintaining the HiSeq method, reducing equipment size and cost, and making operation quicker and easier. MiSeq can produce 3.7 to 4.6 Gb of biometric data within 2 to 150 bp within a maximum of 24 hours.

Currently, NGS is widely used for human whole genome sequence (WGS), exome-seq, RNA-seq, epigenomics research. The genomic analysis based on NGS can be ranked in the order of animal (25%), plant (12%) and microorganism (2%) when 46% is classified as human. In particular, the virus field is only an early stage in utilizing NGS. The reason for this is that microorganisms have a relatively well-conserved sequence of genes, but virus sequences differ in the sequences of individual viruses, and gene mutations occur very frequently, so the research on viruses using NGS is progressing It is slow.

Among the viruses, especially RNA viruses such as Hanta virus, the problem that the sample concentration at the initial stage of infection is very low is the biggest obstacle to discovering WGS of virus using NGS.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a method for easily detecting a target virus (for example, Hanta virus) which is difficult to obtain detection / diagnosis and a whole genome sequence (WGS), and can quickly confirm a full-length genome sequence. As a result, the present inventors have found that a large number of multiplex primer sets for detecting and confirming the full-length genome sequence of a target virus with reference to a reference genome sequence (specifically, a forward direction amplifying target virus genome sequence fragments of 100 to 200 bp long, And a reverse primer pair), designed / manufactured using a mRNA obtained from a sample (for example, blood, serum, urine, etc.) or a cDNA prepared therefrom as a template The present invention has been accomplished by carrying out a polymerase chain reaction (PCR) and confirming by next generation sequencing (NGS) to develop a method for identifying target viruses with high accuracy, sensitivity and reliability.

It is an object of the present invention to provide a method for identifying a full-length genome sequence of a virus of interest in a sample.

Another object of the present invention is to provide a Hanvirus diagnostic kit in a sample.

It is still another object of the present invention to provide a composition for detecting hantavirus-induced diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method for identifying a full-length genome sequence of a virus of interest in a sample comprising the steps of:

(a) preparing a multiplex primer set pool comprising different sets of multiplex primers consisting of a plurality of forward and reverse primer pairs amplifying different 100-200 bp long target virus genomic sequence fragments;

The target viral genomic sequence fragments amplified by the one multiplex primer set include 1 to 2 kb of the target viral genome sequence and two fragments adjacent to each other in the 5'-to-3 'direction in the fragments Wherein the overlapping sequence is common to the 3'-end of the preceding fragment and the 5'-end of the following fragment in a length of 40 to 60 bp;

Wherein all the fragments amplified by the multiplex primer set pool include the full-length genomic sequence of the target virus when overlapping each other in the 5'-to-3 'direction;

(b) performing a multiplex polymerase chain reaction (mPCR) using the prepared primer set pool in the sample; And

(c) analyzing the genomic sequence library containing the amplified mPCR products with next generation sequencing (NGS).

According to another aspect of the present invention, the present invention provides a Hanvirus diagnostic kit in a sample comprising the aforementioned multiplex primer set pool, wherein the multiplex primer set pool comprises a forward direction selected from the group consisting of SEQ ID NOS: 1 to 472 Wherein the primer pair comprises a primer and a reverse primer pair, wherein the primer pair amplifies the genomic sequence in the Hanta virus L, M and S segments.

According to still another aspect of the present invention, there is provided a composition for detecting a hantavirus-induced disease comprising the multiplex primer set pool described above.

The present inventors have sought to develop a method for easily detecting a target virus (for example, Hanta virus) difficult to detect / diagnose and acquire a full-length genome sequence, and to quickly identify a full-length genome sequence. As a result, the present inventors have found that a large number of multiplex primer sets for detecting and confirming the full-length genome sequence of a target virus with reference to a reference genome sequence (specifically, a forward direction amplifying target virus genome sequence fragments of 100 to 200 bp long, And a reverse primer pair), designed / manufactured using a mRNA obtained from a sample (for example, blood, serum, urine, etc.) or a cDNA prepared therefrom as a template A polymerase chain reaction (PCR) was performed and confirmed by next generation sequencing (NGS) to develop methods for identifying target viruses with high accuracy, sensitivity and reliability.

The present invention relates to a method for identifying a target virus (for example, Hanta virus) or a full-length genome sequence thereof by devising / producing a multiplex primer set pool based on a reference genome sequence, wherein said target virus is detected and its fragment genomic sequence It provides a way to quickly acquire the full-length genome sequence at once, rather than confirmation.

According to the present invention, the multiplex primer set pool of the present invention is composed of a plurality of sets of multiplex primer sets which are different from each other, and the set of multiplex primers comprises forward and reverse primers capable of amplifying a part of the genome sequence of the target virus Pairs (step (a)). Specifically, the primer pair in one multiplex primer set is designed to amplify a sequence fragment having a length of 100 to 200 bp, and two fragments adjacent to each other in the 5'-to-3 'direction in the fragments to be amplified Fragments contain an overlapping sequence. The overlapping sequence refers to a complementary sequence to a part of the oligonucleotide sequence of the fragment to be amplified, which is designed to sequentially sequence the two adjacent fragments. More specifically, the nested sequence is common to the 3'-end of the first amplified fragment and the 5'-end of the second amplified fragment in the 5'-to-3 'direction. Therefore, when sequencing fragments of 100 to 200 bp in length amplified by the single multiplex primer set are ligated, it is possible to accurately and quickly secure a partial genome sequence of the target virus. Specifically, the one set of multiplex primers is designed to identify a 1-2 kb genomic sequence of the target viral genome sequence (see Figures 1 and 2).

In some embodiments of the invention, the multiplex primer set of the present invention comprises forward and reverse primer pairs that amplify target virus genomic sequence fragments of 100-200 bp in length, wherein one multiplex primer set comprises a target virus And used to detect a 1-2 kb genomic sequence of the genomic sequence (cf. FIGS. 3 to 5).

In the present invention, the multiplex primer set pool of the present invention, which includes a plurality of different multiplex primer sets, is designed / designed to encompass the full-length genome sequence of the target virus so that all the fragments amplified by the individual multiplex primer set When they are combined and overlapped / linked in the 5'-to-3 'direction, the full-length genomic sequence of the target virus can be obtained.

Then, in the present invention, a multiplex polymerase chain reaction (mPCR) is performed on the sample using the prepared multiplex primer set pool (step (b)).

In some embodiments of the invention, the amplification of the invention is carried out in accordance with a polymerase chain reaction (PCR). In some embodiments of the invention, the primer pairs of the invention are used for amplification reactions.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, traditional PCR procedures have been modified to enhance the specificity or sensitivity of PCR, such as touchdown PCR, hot start PCR, nested PCR and booster PCR, etc. . In addition, multiplex PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction inverse polymerase chain reaction (IPCR), vectorette PCR, and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

The term "amplification reaction" as used herein refers to a reaction that amplifies a target genomic sequence (or nucleic acid molecule). A variety of amplification reactions have been reported in the art, including PCR (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) , Molecular Cloning . A Laboratory Manual , 3rd ed. The method of Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822), multiplex PCR (McPherson and Moller, 2000), ligase chain reaction LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA; WO 88/10315), self- (US Ser. No. 6,410, 276), consensus sequence primed polymerase chain reaction (CP), and the like. US Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification , NASBA; US Including Patent No. 5,130,238, 1 - No. 5,409,818, No. 5,554,517 No., and the No. 6,063,603), and strand substitution amplification (strand displacement amplification), but are not limited to. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and U.S. Patent No. 09 / 854,317. Better still, the amplification process is performed according to the PCR method disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159.

As used herein, the term "primer " means an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid strand (template, template) is induced, that is, polymerization such as nucleotide and DNA polymerase And can act as a starting point for synthesis at the appropriate temperature and pH conditions. Specifically, the primer is a deoxyribonucleotide and a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides. As used herein, the term "oligonucleotide" is a deoxyribonucleotide or ribonucleotide present in single or double stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The appropriate length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer. The term "annealing" or "priming" means that an oligodeoxynucleotide or nucleic acid is apposited to a template (e.g., a genomic sequence or nucleic acid molecule) To form a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.

When the method of the present invention is carried out using a primer, a gene amplification reaction is carried out and the target virus (specifically, Hanta virus) and its full-length genome sequence are simultaneously Detection / confirmation. Therefore, the present invention carries out a gene amplification reaction using a primer pair that binds to cDNA synthesized from the total RNA obtained from the target virus in the sample. To obtain mRNA, total RNA is isolated from the sample. The isolation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al . , Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al . , Plant Mol . Biol . Rep . , ≪ / RTI > 9: 242 (1991); Ausubel, FM et al . , Current Protocols in Molecular Biology , John Willey & Sons (1987); And Chomczynski, P. et al . , Anal . Biochem . 162: 156 (1987)). For example, total RNA can be easily isolated using Trizol. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified.

In some embodiments of the invention, a sample that can be used in the present invention is a sample of blood, serum, sputum, urine or tissue (e.g., lung, kidney, brain, etc.), total RNA obtained therefrom, mRNA or cDNA.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are described in Joseph Sambrook, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

The term "hybridization " as used herein means that two single-stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization can occur either in perfect match between single stranded nucleic acid sequences or in the presence of some mismatching nucleotides. The degree of complementarity required for hybridization can vary depending on hybridization reaction conditions, and can be controlled by temperature. The terms "annealing" and "hybridization" are not different and are used interchangeably herein.

A variety of DNA polymerases can be used in the amplification of the present invention, including the "Clenow" fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Specifically, the polymerase is a thermostable DNA polymerase that can be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , Pyrococcus furiosus (Pfu), Thermus antranikianii , Thermus caldophilus , Thermus chliarophilus, Thermus flavus , Thermus igniterrae , Thermus lacteus , Thermus oshimai , Thermus ruber, Thermus rubens , Thermus scotoductus , Thermus silvanus , Thermus species Z05 , Thermus species sps 17, Thermus thermophilus , Thermotoga maritima , Thermotoga neapolitana And DNA polymerase of Thermosipho africanus .

When performing the polymerization reaction, the reaction vessel may be provided with an excessive amount of components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with such joins as Mg 2 + , dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence (i.e., the genome sequence of the target virus) and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

In addition, the method of the present invention is characterized in that the first multiplex multiplex primer set pool prepared in the step (a) is multiplexed with the multiplex multiplex primer set Th multiplexer primer set pool can be manufactured and amplified and verified in the same manner (e.g., next generation sequencing). In other words, the second multiplex primer set pool is prepared in consideration of the possibility of a site that is not amplified due to a specific point on the nucleotide sequence structure, and the method of the present invention is performed using the second multiplex primer set pool, thereby remarkably increasing the detection specificity and accuracy (See Figures 1 and 3). A second multiplex primer set pool prepared in the same manner as the first multiplex primer set pool consists of multiplexer primer sets of different sequence from the first multiplex primer set pool (Note: Set 2 in FIG. 1) .

In some embodiments of the present invention, the method of the present invention comprises preparing a second multiplex primer set pool comprising multiplex primer sets comprised of a primer pair different from the multiplex primer set pool prepared in step (a) (Step (b-1); Fig. 3).

In addition, the present invention can repeatedly confirm the detection of the target virus and its acquisition of the full-length genome sequence by performing a secondary PCR amplification reaction using the primary PCR amplification product as a template (see FIG. 5).

Finally, the present invention analyzes the amplification products of amplified genomic sequence fragments (for example, amplification fragments for L, M or S fragments of Hanta viruses) as described above in a suitable manner to detect the genomic sequence of the desired virus ( Step (c)). For example, after performing the above-described amplification reaction by gel electrophoresis and observing and analyzing the resultant bands to confirm presence or absence thereof, the genome sequence of the target virus can be easily identified through next generation sequencing (for example, NGS equipment) (Refer to FIGS. 6 to 8).

Accordingly, the present invention also provides a hepatitis virus diagnostic kit in a sample comprising the aforementioned multiplex primer set pool. The diagnostic kit of the present invention is characterized in that the multiplex primer set pool (set 1 or set 2) comprises a forward primer and a reverse primer pair selected from the group consisting of SEQ ID NOS: 1 to 472, , M and S segment genomic sequences. In some embodiments of the invention, the L segment of hanta virus (specifically, A. a_10-434 strain) in the multiplex primer set pool comprises a forward primer selected from the group consisting of SEQ ID NOS: 1 to 130 and a reverse primer pair Or using a forward primer and a reverse primer pair selected from the group consisting of SEQ ID NOS: 131 to 260; The M segment of hanta virus (specifically, A.a_10-434 strain) comprises a forward primer selected from the group consisting of a forward primer and a reverse primer selected from the group consisting of SEQ ID NOS: 261 to 332, or a forward primer selected from the group consisting of SEQ ID NOS: 333 to 404, Amplified using a reverse primer pair; The S segment of hanta virus (specifically, A. a_10-434 strain) comprises a forward primer selected from the group consisting of a forward primer and a reverse primer selected from the group consisting of SEQ ID NOS: 405 to 438 or a forward primer selected from the group consisting of SEQ ID NOS: 439 to 472, And amplified using a reverse primer pair.

In addition, the present invention provides a composition for detecting a hantavirus-induced disease comprising the above multiplex primer set pool.

The methods and kits of the present invention can be applied to any known virus having a full-length genomic sequence, including, for example, hanta virus. Specifically, the method of the present invention is a method for diagnosing and obtaining the full-length genomic sequence of Hanta virus (i.e., conventional or novel hanta virus) in which the reference full-length genomic sequence is known, It makes it very easy to acquire the accuracy, sensitivity, reliability and full-length genome sequence (WGS) for viruses. When using the multiplex primer set prepared in the present invention, the detection of hantavirus and its full-length genome sequence with very high accuracy, sensitivity, and reliability could be determined (see FIG. 8).

In some embodiments of the present invention, the target virus of the present invention is a hanta virus, and more specifically, hanta virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), PUU virus , Sin Nombre virus (SNV), Black Creek Canal virus (BCCV), Bayou virus (BAYV), New York virus (NYV), Andes virus (ANDV) It may be the Laguna Negra virus (LNV), more particularly the hanthan virus.

In some embodiments of the invention, the huntervirus-induced diseases detectable by the composition of the invention are selected from the group consisting of hemorrhagic fever with renal syndrome (HFRS), Hantavirus cardiopulmonary syndrome (HCPS), nephropathia epidemica, hemorrhagic fever caused by Seoul virus, sweating sickness, Crimean-Congo hemorrhagic fever (CCHF), La Crosse encephalitis, Phlebotomus fever, And Rift Valley fever, and more specifically include HFRS and HCC virus.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method for obtaining a hapta virus full-length genome sequence using multiplex PCR-based next generation sequencing and its use.

(b) The methods, kits and compositions of the present invention utilize a multiplex primer set pool consisting of a set of multiplex primers comprising forward and reverse primer pairs capable of amplifying some genomic sequences of the desired virus, The presence or absence of a target virus can be confirmed easily and quickly.

(c) In addition, two fragments adjacent to each other in the 5'-to-3 'direction in the 100 to 200 bp long sequence fragments obtained by the primer pairs included in the multiplex primer set have an overlapping sequence, . Accordingly, when the fragments amplified by the multiplex primer set pool are all collected and overlapped / linked in the 5'-to-3 'direction, the full-length genome sequence of the target virus can be easily obtained.

(d) Thus, the methods, kits and compositions of the present invention allow very precise and rapid acquisition of the full-length genomic sequence of a target virus (e.g., Hanta virus) with high-depth coverage, It can be more effectively applied to diagnosis of a virus-induced disease.

Brief Description of the Drawings Fig. 1 is a schematic diagram of a multiplex primer for specifically amplifying a hanta virus whole genome sequence used in the present invention. Fig. Two types of multiplex primer set 1 and set 2 were designed. Multiplex primer set 1 designed primer pairs so that the gene can be amplified by a length of 150 bp in each segment of Hanta virus L, M, S. Considering the possibility that the nucleotide sequence may not be amplified due to structural specificities, an overlapping region of 50 bp overlapping between the primers was selected. Also, in order to increase the detection specificity in consideration of the case where the primer did not amplify or the base sequence was not read properly in some gene sections, the multiplex primer set 2 was designed in the same way from the set 25 bp away from the set 1 . And each set included a primer OSM55 (5'-TAGTAGTAGACTCC-3 '; SEQ ID NO: 473) that specifically binds to each conserved region.
FIG. 2 is a diagram illustrating the expected size of a product when PCR was performed using the multiplex primer set 1 (or set 2) designed in FIG. An approximately 250 bp product can be generated that is generated by combining a PCR product of about 150 bp in length, such as A or B as shown in Figure 2, as well as a reverse primer that is about 100 bp away from the reverse primer of A, such as C. In the same way, the size of the product increases every 100 bp, and the shape of the whole product is expected to appear like a ladder shape. That is, products of varying sizes may be present between 150 bp and 1 kbp. Depending on the amount and condition of the template DNA used in the PCR, the polymer chain reaction product may appear as a ladder-like appearance and a dark or cloudy appearance.
FIG. 3 is an experiment using the multiplex primer of FIG. 1 produced by the method of the present invention. In FIG. 3, the multiplex primer sets 1 and 2 corresponding to the Hanta virus L, M, or S segments in two isolate strains, 2 (PCR product) (1 st PCR product). It can be seen that the appearance of the reaction product is in the shape of a ladder as expected in Fig.
FIG. 4 is a PCR experiment using the multiplex primer set of FIG. 1 designed and manufactured by the method of the present invention, showing that multiplex primers for each of the Hanta virus L, M, or S fragments in five species of lung tissue The results of polymerase chain reaction (1 st PCR product) using Set 1 are shown.
FIG. 5 shows an experiment using the multiplex primer of FIG. 1 designed and constructed according to the present invention, in which 15 sets of patient samples were subjected to multiplex primer set 1 corresponding to Hantavirus L, M or S fragments The PCR product was diluted with an appropriate amount and further subjected to secondary PCR (2 nd PCR product).
Figure 6 compares consensus PCR with SISPA-NGS to compare the accuracy and homology of the multiplex-based NGS results. As a result of analyzing the consensus PCR, the multiplexed NGS compared to SISPA-NGS (Horizontal axis, reference object; and vertical, objective sample).
FIG. 7 is a graph showing the results of the SISPA (Sequence Independent, Single Primer Amplification; Molecular and Cellular Probes Volume 5, Issue 6, December 1991, Pages 473-416) method, which is generally used to obtain the full-length genome sequence (WGS) And mapping the FASTQ file, which is the result of the reaction with MiSeq, an NGS device, to each L, M, or S segment sequence using the reference genome sequence of the Hanta virus. As shown in FIG. 7, the distribution at the distal end of each segment is lower than that of the other segments, and a gap is generated in the L segment having a longer length than the other segments.
FIG. 8 is a graph showing a result of the pre-treatment of a sample using a multiplex primer set pool designed and manufactured according to the present invention, and a FASTQ file as a result of the reaction with MiSeq, an NGS instrument, L, M, or S segment sequence. As shown in FIG. 8, the overall distribution of each segment was high and uniform, and no gap occurred at all, indicating that the whole genome sequence of the Hanta virus can be obtained at one time.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

1. Multiplex for detection of whole genome of hanta virus Primer  Design and production

In order to design and construct a primer set capable of specifically detecting Hanta virus, a full-length genome sequence of the Hantaan virus strain belonging to the Hanta virus family A.a_10-434 is referred to as a reference genome sequence (Reference Genome Sequence: Sequence A.a_10-434, which has its own sequence).

FIG. 1 is a schematic diagram of a multiplex primer for amplifying the whole genome sequence of the hanta virus used in the present invention into hanta virus specific fragments. Two types of multiplex primer set 1 and set 2 were designed. Multiplex primer set 1 was designed so that the gene could be amplified by a length of 150 bp in each of L, M or S segments of Hanta virus. Considering the possibility that the nucleotide sequence may not be amplified due to the structural specificity, sites overlapping by 50 bp between the primers were selected. Also, in order to increase the detection specificity in consideration of the case where the primer did not amplify or the base sequence was not read properly in some gene sections, the multiplex primer set 2 was designed in the same way from the set 25 bp away from the set 1 . In each set, a primer OSM55 specific to each segmental end was included. The primer sequences of the multiplex primer sets 1 and 2 of the hanta virus L, M and S fragments prepared in the present invention are shown in Tables 1 and 2, Tables 3 and 4, and Tables 5 and 6, respectively.

FIG. 2 is a diagram illustrating the expected product size when PCR was performed using the multiplex primer set 1 (or set 2) designed in FIG. A product of about 250 bp, which is generated by combining with a reverse primer that is about 100 bp away from the reverse primer of A, such as C, as well as a PCR product of about 150 bp in length, as shown in Fig. In the same way, the size of the product increases every 100 bp, and the shape of the whole product is expected to appear like a ladder shape. That is, products of varying sizes may occur between 150 bp and 1 kbp. Depending on the amount and condition of the template DNA used in the PCR, the polymer chain reaction product may appear as a ladder-like, dark or cloudy appearance.

Multiplex primer sequence for Hanta virus L segment identification (set 1). Multiplex primer sequence set for L segment identification 1 OligoID Sequences (5 '-> 3') HNL01F TAGTAGTAGACTCCGGAAGT HNL02F GTAGAGTGTATAGATTATCT HNL03F AGGCAATAGGGAAAGTACTA HNL04F CAAGGCCTTTTTCAAGATGA HNL05F AGAGAAAAAGAAGCTTAAATA HNL06F CATTTAATGTAGTGGCAGTT HNL07F AGCTGAGATAAGTTACGTTA HNL08F AGCCAGCCATATTACATACC HNL09F GAAATTTTCATTTTTTGAA HNL10F TAGAAACTTCTTACTAATAC HNL11F CTGAACTTAATTCCAGATAC HNL12F CACGAATACAATTTAAACAG HNL13F ACTCAATAATCATAAAAGTG HNL14F AAAATATTGTCTGATTTAGA HNL15F TTCAGAATGAGACCCAGAAG HNL16F TCATTCAGGATTAAAAAGAT HNL17F GGTTCCTTTATTAGGTTCAT HNL18F GGGTATCCAAAGTCATGAGC HNL19F TACAGAAGACCAAGGGCAGT HNL20F ATTTTTGACAATCTACGTTA HNL21F CATTAGAGGTATATATATAT HNL22F AACAGTGGACCAGTCGACTG HNL23F ACCTGTTTCTTTTTATTTGA HNL24F AGGAAGAAAAGTATGGGGAA HNL25F TGCTGTTGAGTTGGCAGCAA HNL26F TTTAGCCAAACAAGAAATAT HNL27F ATTTATCAAATTCAAGACAT HNL28F AGAGGCAGATAGGGGCTTTT HNL29F GAGTACATATCATATGGAGG HNL30F GTAATCATAAATTCATTAGG HNL31F TACCTCCATGCTACATAATG HNL32F AAACTAAGGAACTATATTGA HNL33F ACTGGCTGCAGGGTAACTTA HNL34F TTGCTTTTTTGAGTTTGCAC HNL35F CAACAGATTCAAGCAGGCCA HNL36F TTAAGATCTCACCAAAGAAA HNL37F ACTCTTAGGTTCTCTGTCAG HNL38F CCTCAGGTTGCACAACTAGC HNL39F AAGTTAAGCATGCTGATACA HNL40F ATTAAAGCGCGCACTTCTAG HNL41F TTTCAACATGAGAGGTTAGG HNL42F CAAAATTTTTAGAGTTATGG HNL43F TGACCCAAGTATTGTTACAG HNL44F GAATGGGTTACCTTCAGGGA HNL45F GTTGTACATTTTCTAAAGAG HNL46F AACATTCACAGTCAGAGAAA HNL47F CTGGATACAGCCAAGTTCCC HNL48F TTATGAAAAGAATTGCACCT HNL49F CTGGCTGAAATCAATGTCAC HNL50F CAACAGAAAGACTTAGCTGC HNL51F TCGAGGATAAGACCTTTTCA HNL52F TATAAAGGGTGATTATTCAG HNL53F ACAAAGCTTTATGAAGGAGA HNL54F CATCAATTACACAGAAGCAT HNL55F AGTACGGCACATGGTATTGT HNL56F CTTAGAACCACACAGGCAAT HNL57F TATTAGCTCATGCGTTTCAT HNL58F AAATCCCATCATTTCATCTG HNL59F ATATCTGTTGATCTCTTTAT HNL60F GTACTGTGAGTTCAATGTCA HNL61F TGTGGATTTTGAGAATATAG HNL62F GAATCAAAGAAAATGAGGGG HNL63F CTGTTAATATTATCTTAATG HNL64F GAGCATTGTCCGAGGATGGG HNL65F GATGTCATTCCTGATTCATT HNL01R ATGTCATGTCTCACGGCGTA HNL02R TATAATGTTTGAAGGGACCC HNL03R TTGTTCCATTAATTTTATAA HNL04R TCTTGTTCAATATAAGCTAA HNL05R TTGTGTAGTAATATTGGAGC HNL06R CTCTCTCCTCAGTTCTGATC HNL07R GCTCCTATCGGTTCCATCCC HNL08R AAAGCACTCGAACACTGCTG HNL09R CAGGTTTGTAAGATGTGATT HNL10R GCCATGTCATGGATCAGGTA HNL11R AAATGTTCCTGGCTCTCTTA HNL12R GACTCTCTATTTGCGCACCA HNL13R TCTTGAGTTGATGGCTCAGT HNL14R TGTTGTCTTTTTCAACACAT HNL15R TATTGTATGAGTGTAAGGAC HNL16R ACTAAACCAGGTCCTATCCT HNL17R ATTTAATGCTAGCAGCCTAT HNL18R ACACAGATCTAATTGCATAT HNL19R GAGTATAATGATGTCACTGC HNL20R AAGTGCAACTAACAAGCTTT HNL21R TAAATGAAGGGTAAACTCCA HNL22R TCTTCATTCATATTTCCATG HNL23R CCACATCATATATCCATTTT HNL24R ACTTTGTAGCCAGGACTTTA HNL25R TGAACCTGTCCTGACATACC HNL26R CTCCTCATATAATTTTAAGA HNL27R GCCTGCATCTAGTAGGAAGT HNL28R GCCCCTTGGATTGCAAGAAT HNL29R TGCACTAACATACATGAGTT HNL30R CACAGTTCTTTAGCTTATTG HNL31R TTGATGTGTGGGTCAAGGCT HNL32R TGCAACACCGAAAAGCGAAG HNL33R CATAAATGAATAATGCATCG HNL34R ATCTCTGTATTTACACTAAA HNL35R AAATTCAGCATTTGTCGGGG HNL36R AATCGTCAAAATAACCTAAA HNL37R CTTTCAACTTTACTGGTGCA HNL38R CATTGCACCATTTCCTCCCA HNL39R GCATTGATTTCTGTCTCTTG HNL40R CACTGTACTTTCCCAATAAA HNL41R AATATAATCATAAGTTACAT HNL42R GGAGCTGCAAGGGTGACTGC HNL43R TCTGCAAAACTGTTAGCAGC HNL44R AATTCCATTCAAGAAATCTT HNL45R CAGGAATACTATTTTGTATG HNL46R ATTGTCTTTAAGTCAACAGA HNL47R TACCCTTGATTTAGAAGATT HNL48R TATGAGGCTTCACTCTTATA HNL49R CATCTCCAAATTTCTATGCA HNL50R CCCAACCCTACAGAACCGAT HNL51R ATGCTCGCCTGTTGGCTTGT HNL52R TCAAGACCAAAATTGAATGT HNL53R CGAAACCTCATCCATCATGA HNL54R TCCCCCATTCAAATCCAGAC HNL55R TCCTTTTTGTAATCATAAA HNL56R TACTGTTTGATACCTAATAT HNL57R GAAGATTCTTCATAAAATTC HNL58R GGGTTAATCAGCTTAAGTAA HNL59R TAAGCTCATTTCTTCAGACC HNL60R GTAGGAAATGTTCAATATCT HNL61R AGTCTGACAGGCTCAAGTAT HNL62R CAAATTAAATGTTTTAGAGA HNL63R GATTTTCAAACTGATCAATA HNL64R AACAGTACCATGGTATGCCT HNL65R TTAGTAGTAGGTATGCTCCG

Multiplex primer sequence for Hanta virus L segment identification (set 2). Multiplex for L segment identification primer  Sequence set 2 OligoID Sequences (5 '-> 3') HNL01F TAGTAGTAGACTCCGGAAGT HNL02F GTAGAGTGTATAGATTATCT HNL03F AGGCAATAGGGAAAGTACTA HNL04F CAAGGCCTTTTTCAAGATGA HNL05F AGAGAAAAAGAAGCTTAAATA HNL06F CATTTAATGTAGTGGCAGTT HNL07F AGCTGAGATAAGTTACGTTA HNL08F AGCCAGCCATATTACATACC HNL09F GAAATTTTCATTTTTTGAA HNL10F TAGAAACTTCTTACTAATAC HNL11F CTGAACTTAATTCCAGATAC HNL12F CACGAATACAATTTAAACAG HNL13F ACTCAATAATCATAAAAGTG HNL14F AAAATATTGTCTGATTTAGA HNL15F TTCAGAATGAGACCCAGAAG HNL16F TCATTCAGGATTAAAAAGAT HNL17F GGTTCCTTTATTAGGTTCAT HNL18F GGGTATCCAAAGTCATGAGC HNL19F TACAGAAGACCAAGGGCAGT HNL20F ATTTTTGACAATCTACGTTA HNL21F CATTAGAGGTATATATATAT HNL22F AACAGTGGACCAGTCGACTG HNL23F ACCTGTTTCTTTTTATTTGA HNL24F AGGAAGAAAAGTATGGGGAA HNL25F TGCTGTTGAGTTGGCAGCAA HNL26F TTTAGCCAAACAAGAAATAT HNL27F ATTTATCAAATTCAAGACAT HNL28F AGAGGCAGATAGGGGCTTTT HNL29F GAGTACATATCATATGGAGG HNL30F GTAATCATAAATTCATTAGG HNL31F TACCTCCATGCTACATAATG HNL32F AAACTAAGGAACTATATTGA HNL33F ACTGGCTGCAGGGTAACTTA HNL34F TTGCTTTTTTGAGTTTGCAC HNL35F CAACAGATTCAAGCAGGCCA HNL36F TTAAGATCTCACCAAAGAAA HNL37F ACTCTTAGGTTCTCTGTCAG HNL38F CCTCAGGTTGCACAACTAGC HNL39F AAGTTAAGCATGCTGATACA HNL40F ATTAAAGCGCGCACTTCTAG HNL41F TTTCAACATGAGAGGTTAGG HNL42F CAAAATTTTTAGAGTTATGG HNL43F TGACCCAAGTATTGTTACAG HNL44F GAATGGGTTACCTTCAGGGA HNL45F GTTGTACATTTTCTAAAGAG HNL46F AACATTCACAGTCAGAGAAA HNL47F CTGGATACAGCCAAGTTCCC HNL48F TTATGAAAAGAATTGCACCT HNL49F CTGGCTGAAATCAATGTCAC HNL50F CAACAGAAAGACTTAGCTGC HNL51F TCGAGGATAAGACCTTTTCA HNL52F TATAAAGGGTGATTATTCAG HNL53F ACAAAGCTTTATGAAGGAGA HNL54F CATCAATTACACAGAAGCAT HNL55F AGTACGGCACATGGTATTGT HNL56F CTTAGAACCACACAGGCAAT HNL57F TATTAGCTCATGCGTTTCAT HNL58F AAATCCCATCATTTCATCTG HNL59F ATATCTGTTGATCTCTTTAT HNL60F GTACTGTGAGTTCAATGTCA HNL61F TGTGGATTTTGAGAATATAG HNL62F GAATCAAAGAAAATGAGGGG HNL63F CTGTTAATATTATCTTAATG HNL64F GAGCATTGTCCGAGGATGGG HNL65F GATGTCATTCCTGATTCATT HNL01R ATGTCATGTCTCACGGCGTA HNL02R TATAATGTTTGAAGGGACCC HNL03R TTGTTCCATTAATTTTATAA HNL04R TCTTGTTCAATATAAGCTAA HNL05R TTGTGTAGTAATATTGGAGC HNL06R CTCTCTCCTCAGTTCTGATC HNL07R GCTCCTATCGGTTCCATCCC HNL08R AAAGCACTCGAACACTGCTG HNL09R CAGGTTTGTAAGATGTGATT HNL10R GCCATGTCATGGATCAGGTA HNL11R AAATGTTCCTGGCTCTCTTA HNL12R GACTCTCTATTTGCGCACCA HNL13R TCTTGAGTTGATGGCTCAGT HNL14R TGTTGTCTTTTTCAACACAT HNL15R TATTGTATGAGTGTAAGGAC HNL16R ACTAAACCAGGTCCTATCCT HNL17R ATTTAATGCTAGCAGCCTAT HNL18R ACACAGATCTAATTGCATAT HNL19R GAGTATAATGATGTCACTGC HNL20R AAGTGCAACTAACAAGCTTT HNL21R TAAATGAAGGGTAAACTCCA HNL22R TCTTCATTCATATTTCCATG HNL23R CCACATCATATATCCATTTT HNL24R ACTTTGTAGCCAGGACTTTA HNL25R TGAACCTGTCCTGACATACC HNL26R CTCCTCATATAATTTTAAGA HNL27R GCCTGCATCTAGTAGGAAGT HNL28R GCCCCTTGGATTGCAAGAAT HNL29R TGCACTAACATACATGAGTT HNL30R CACAGTTCTTTAGCTTATTG HNL31R TTGATGTGTGGGTCAAGGCT HNL32R TGCAACACCGAAAAGCGAAG HNL33R CATAAATGAATAATGCATCG HNL34R ATCTCTGTATTTACACTAAA HNL35R AAATTCAGCATTTGTCGGGG HNL36R AATCGTCAAAATAACCTAAA HNL37R CTTTCAACTTTACTGGTGCA HNL38R CATTGCACCATTTCCTCCCA HNL39R GCATTGATTTCTGTCTCTTG HNL40R CACTGTACTTTCCCAATAAA HNL41R AATATAATCATAAGTTACAT HNL42R GGAGCTGCAAGGGTGACTGC HNL43R TCTGCAAAACTGTTAGCAGC HNL44R AATTCCATTCAAGAAATCTT HNL45R CAGGAATACTATTTTGTATG HNL46R ATTGTCTTTAAGTCAACAGA HNL47R TACCCTTGATTTAGAAGATT HNL48R TATGAGGCTTCACTCTTATA HNL49R CATCTCCAAATTTCTATGCA HNL50R CCCAACCCTACAGAACCGAT HNL51R ATGCTCGCCTGTTGGCTTGT HNL52R TCAAGACCAAAATTGAATGT HNL53R CGAAACCTCATCCATCATGA HNL54R TCCCCCATTCAAATCCAGAC HNL55R TCCTTTTTGTAATCATAAA HNL56R TACTGTTTGATACCTAATAT HNL57R GAAGATTCTTCATAAAATTC HNL58R GGGTTAATCAGCTTAAGTAA HNL59R TAAGCTCATTTCTTCAGACC HNL60R GTAGGAAATGTTCAATATCT HNL61R AGTCTGACAGGCTCAAGTAT HNL62R CAAATTAAATGTTTTAGAGA HNL63R GATTTTCAAACTGATCAATA HNL64R AACAGTACCATGGTATGCCT HNL65R TTAGTAGTAGGTATGCTCCG

Multiplex primer sequence (set 1) for identification of the Hanta virus M segment. Multiplex for identification of H-virus M segment primer  Sequence set 1 OligoID Seq  (5 ' - > 3 ') HNM01F TAGTAGTAGACTCCGCAAAATAAAG HNM02F AATGTCTATGACATGAAAAT HNM03F CAGCACAGCTGGTGCCTGAG HNM04F TCAGTCACAGTCTAGCCAAA HNM05F CGCAGTAGAAAATCAATAAC HNM06F TGAAAAGCTGTTTGATTGCA HNM07F CCCAGATCAGAGTGTGGTCA HNM08F AAAATTTTTGAACAGGTTAA HNM09F CACCAATTTATGTTCCAACA HNM10F AGAAACTGCAACTTACTCTA HNM11F TCTTATTCTTCCCTTAGTAT HNM12F GTGCCATACCACTCATGTGG HNM13F ATCATGTGAGGCCTTTTCTG HNM14F AATTTTGTATGTCAGCGAGT HNM15F ACACTATAACAAGCTTATTT HNM16F GCTTGTTACATTCTGTTTTG HNM17F CAAGAAAACAGGTTAAAATC HNM18F CCTATAAAGAGTTGAAGGCA HNM19F TTACAAAGTATGCCAAGTTA HNM20F TTTAGATACAAAAGTAGGTG HNM21F CTCCTGTCTGGAATGACAAT HNM22F CCGTAGGAAGCTAACAAACC HNM23F CACTGGTTTGATGGTCGTCT HNM24F AAAGGGATTACCAATATGAG HNM25F GAAACCAGTTGGCAGTGCTT HNM26F AATGACTGTTTTGTATCTAG HNM27F GTGGCGGTCTAATATTTAAA HNM28F GTTCCCAGGTAGTTTCAGGA HNM29F ATTGATTCCTTTCAATCTTT HNM30F TCTTAGTAACAAAAGACATC HNM31F AGGGTTTACATTAACATGTC HNM32F GTAACATTAACAAGAGGACA HNM33F TTGGACTCCATGCTGCTGCA HNM34F AAAATGTTGGTTTGTTAAAT HNM35F GTTTTACTGAGCATTCTCTG HNM36F TTTTTATATTCCAGTATAAT HNM01R TCCCCGAAGCTTACTGTGTG HNM02R TGATTGGTGATTATCCATGC HNM03R CAACTTCAGTAGACACTGTC HNM04R TAAGTACTATTGCATGATAG HNM05R ATAAACTACCTGTACTCTGT HNM06R CGATATCAAAGATCCCATGC HNM07R GTATCATTGCATGTTGATTC HNM08R GAATGCTTCCATGGATCTGA HNM09R GAGGAACTTTTGCATTGGCA HNM10R ACATGTTTGGCTTCTGTTGA HNM11R ATAATATCCAGGTAAATCAA HNM12R TGGGGGAGGTTATGTTGAAA HNM13R CCATTGCAGTACACAACGAT HNM14R AATAGAGTGTGCTACCCCAG HNM15R TAAATGTGATTGCTGGTATA HNM16R TCAAACTCTTCCTTTATCTT HNM17R GCATTGAGACTGGGGGCATG HNM18R TTTTCTTTAGATCATCCCTG HNM19R AGAAGAAATATCCACATTGT HNM20R GTGCATAGGAACAGAACCCA HNM21R GTAGATCAATGGATTGTGCT HNM22R CCATAACAGTGAAAGGATGT HNM23R ACAATCTGATGGATTACAAC HNM24R TCCTGCTATACCTTATTGTG HNM25R ATTGTACCAATTATACAGAC HNM26R GCCAAATTGACATGTGGATG HNM27R AAATAGGAGTAGTAGCAAAG HNM28R TCATCAGTAAAGTGCATAGT HNM29R GCAAGGATTTTCACCAAGGT HNM30R AAAAGGTAGGACATTCTGTT HNM31R CCACCTTTTCCTGACACCTT HNM32R AGAAATCCCATTGACCTTGT HNM33R CACTGAATATCCCTGAAATC HNM34R AGCTATGACTTTTTATGCTT HNM35R GCTACTATTTTTTTTTAGTG HNM36R TAGTAGTAGACTCCGCAAAATGTTA

Multiplex primer sequence (set 2) for identification of the Hanta virus M segment. Multiplex for identification of H-virus M segment primer  Sequence set 2 OligoID Seq  (5 ' - > 3 ') HNM01F TAGTAGTAGACTCCGCAAAATAAAG HNM02F AATGTCTATGACATGAAAAT HNM03F CAGCACAGCTGGTGCCTGAG HNM04F TCAGTCACAGTCTAGCCAAA HNM05F CGCAGTAGAAAATCAATAAC HNM06F TGAAAAGCTGTTTGATTGCA HNM07F CCCAGATCAGAGTGTGGTCA HNM08F AAAATTTTTGAACAGGTTAA HNM09F CACCAATTTATGTTCCAACA HNM10F AGAAACTGCAACTTACTCTA HNM11F TCTTATTCTTCCCTTAGTAT HNM12F GTGCCATACCACTCATGTGG HNM13F ATCATGTGAGGCCTTTTCTG HNM14F AATTTTGTATGTCAGCGAGT HNM15F ACACTATAACAAGCTTATTT HNM16F GCTTGTTACATTCTGTTTTG HNM17F CAAGAAAACAGGTTAAAATC HNM18F CCTATAAAGAGTTGAAGGCA HNM19F TTACAAAGTATGCCAAGTTA HNM20F TTTAGATACAAAAGTAGGTG HNM21F CTCCTGTCTGGAATGACAAT HNM22F CCGTAGGAAGCTAACAAACC HNM23F CACTGGTTTGATGGTCGTCT HNM24F AAAGGGATTACCAATATGAG HNM25F GAAACCAGTTGGCAGTGCTT HNM26F AATGACTGTTTTGTATCTAG HNM27F GTGGCGGTCTAATATTTAAA HNM28F GTTCCCAGGTAGTTTCAGGA HNM29F ATTGATTCCTTTCAATCTTT HNM30F TCTTAGTAACAAAAGACATC HNM31F AGGGTTTACATTAACATGTC HNM32F GTAACATTAACAAGAGGACA HNM33F TTGGACTCCATGCTGCTGCA HNM34F AAAATGTTGGTTTGTTAAAT HNM35F GTTTTACTGAGCATTCTCTG HNM36F TTTTTATATTCCAGTATAAT HNM01R TCCCCGAAGCTTACTGTGTG HNM02R TGATTGGTGATTATCCATGC HNM03R CAACTTCAGTAGACACTGTC HNM04R TAAGTACTATTGCATGATAG HNM05R ATAAACTACCTGTACTCTGT HNM06R CGATATCAAAGATCCCATGC HNM07R GTATCATTGCATGTTGATTC HNM08R GAATGCTTCCATGGATCTGA HNM09R GAGGAACTTTTGCATTGGCA HNM10R ACATGTTTGGCTTCTGTTGA HNM11R ATAATATCCAGGTAAATCAA HNM12R TGGGGGAGGTTATGTTGAAA HNM13R CCATTGCAGTACACAACGAT HNM14R AATAGAGTGTGCTACCCCAG HNM15R TAAATGTGATTGCTGGTATA HNM16R TCAAACTCTTCCTTTATCTT HNM17R GCATTGAGACTGGGGGCATG HNM18R TTTTCTTTAGATCATCCCTG HNM19R AGAAGAAATATCCACATTGT HNM20R GTGCATAGGAACAGAACCCA HNM21R GTAGATCAATGGATTGTGCT HNM22R CCATAACAGTGAAAGGATGT HNM23R ACAATCTGATGGATTACAAC HNM24R TCCTGCTATACCTTATTGTG HNM25R ATTGTACCAATTATACAGAC HNM26R GCCAAATTGACATGTGGATG HNM27R AAATAGGAGTAGTAGCAAAG HNM28R TCATCAGTAAAGTGCATAGT HNM29R GCAAGGATTTTCACCAAGGT HNM30R AAAAGGTAGGACATTCTGTT HNM31R CCACCTTTTCCTGACACCTT HNM32R AGAAATCCCATTGACCTTGT HNM33R CACTGAATATCCCTGAAATC HNM34R AGCTATGACTTTTTATGCTT HNM35R GCTACTATTTTTTTTTAGTG HNM36R TAGTAGTAGACTCCGCAAAATGTTA

Multiplex primer sequence for Hanta virus S segment identification (set 1). Multiplex for checking the S-segment of Hanta virus primer  Sequence set 1 OligoID Seq  (5 ' - > 3 ') HNS01F TAGTAGTAGGCTCCCTAAAG HNS02F GGCAGAAGGTAAGGGATGCA HNS03F GGCAAAGATTGATGAGTTAA HNS04F CATCTGAAAGAGAGATCAAT HNS05F TTGTTGTCTATCTTACATCC HNS06F TCGGATTCGATTTAAGGATG HNS07F AAGGCAGAAGAGATTACACC HNS08F TGATTGGTTTCCTGGCATTG HNS09F CCTTGGTGGTCCTGCAACAA HNS10F GAAGCAGCTGGCTGTAGCAT HNS11F TAGCAGGTATTGCTGAGCTT HNS12F GCGAAAGAAATCATCATTCT HNS13F GGAAAGGAGGCTGTGGACAA HNS14F CCAACCAAGAGCCTTTAAAA HNS15F TTATCAGGGGAATCAGTATA HNS16F AGGCTGCCTTAAGTAGCCTT HNS17F TACTAACAACAACACTCTAC HNS01R CTCATCTGGATCCTTTCCAT HNS02R CAGTTGCAATCCTATCTGCC HNS03R TTTAAATCCAGCACATTACC HNS04R TAGAGCTTTCAGAAGTATCG HNS05R GGATACCGTTAACGTCCTCG HNS06R AGTCCACAGACTGCTGTTCT HNS07R TTGTTCAATACGATCACTCC HNS08R CCACTTGCCGCTGCCGTAAG HNS09R ATTGATGATGGTGACTCAAT HNS10R CATGTCCTGCAGGATGGAAA HNS11R TTGATTGTGTCCTTCTGAGA HNS12R TCAGGATCCATATCATCCCC HNS13R TAAAAGGATTAATACATTCA HNS14R TTCCCACCCATAAATGTTCC HNS15R AAATGAAATCTACATCCATA HNS16R GTAGTTAAGTTGAGGTAGTT HNS17R GTAGTAGTTTGCTCCCTAA

Multiplex primer sequence (Set 2) for Hanta virus S segment identification. Multiplex for checking the S-segment of Hanta virus primer  Sequence set 2 OligoID Seq  (5 ' - > 3 ') HN2S01F CTAGAACAACGATGGCAACT HN2S02F ACAGTATGGAAAGGATCCAG HN2S03F CAGCTGGCAGATAGGATTGC HN2S04F GTTATGGTAATGTGCTGGAT HN2S05F TGTCCCGATACTTCTGAAAG HN2S06F TCGTTCGAGGACGTTAACGG HN2S07F GATATAGAACAGCAGTCTGT HN2S08F GGATTGGAGTGATCGTATTG HN2S09F GACTACTTACGGCAGCGGCA HN2S10F AGGACATTGAGTCACCATCA HN2S11F ATTTTTTCCATCCTGCAGG HN2S12F TCTTATCTCAGAAGGACACA HN2S13F ACTTAGGGGATGATATGGAT HN2S14F ATCAATGAATGTATTAATCC HN2S15F GCAAGGGAACATTTATGGGT HN2S16F GTATATATGGATGTAGATTT HN2S17F CACAAAACTACCTCAACTTA HN2S01R GGTCTGTTAATGCTCTCTTG HN2S02R TCTTGTTCCTTCCCAAGGTT HN2S03R TGTGGGTTCATCAATATCCA HN2S04R GCCTCCCCCTTGTTGTCAAC HN2S05R GACACATAAAGATGTTTTGG HN2S06R CCGTGCCTTGATCTGTGCAG HN2S07R GGAGCTTGCAAGGTTCGCTT HN2S08R TCTTTTGTCTCCATATTGCC HN2S09R GTCTGGTGCTCCAGCAAATA HN2S10R TCTTAGATGCCATGATTGTA HN2S11R ATCCTTTGGTCCAGTTGTAT HN2S12R CAAGCTCTGTGCAAGTGTTC HN2S13R GATTCAGTAGTATATGATAA HN2S14R TAACTGACCCACCCCTGAGT HN2S15R AAGAAAGCAAGATTAGTTAA HN2S16R GACAATCAAGGAGCAATCAA HN2S17R GTAGTAGTTTGCTCCCTAA

2. Multiplex for identification of viruses from patient samples primer  Polymerization chain reaction

2-1. Sampling and RNA  extraction

Serum samples were obtained from patients with suspected renal haemorrhagic fever (HFRS). Total RNA was isolated from the obtained serum samples using TRIzol (Invitrogen).

2-2. cDNA  synthesis

1 μg of isolated total RNA was mixed with 2 μl of the OsM55 primer specific to the hanta virus genome, 100 units of M-MLV reverse transcriptase, 0.5 mM of 1 × RT buffer (Promega), 20 units of RNase inhibitor and 20 μl of final volume. The mixture was reacted at 37 ° C for 10 minutes, reacted at 42 ° C for 50 minutes, and reacted at 94 ° C for 3 minutes to synthesize cDNA.

2-3. Multiplex Primary PCR  Reaction and confirmation

3.0 μl of the obtained template cDNA and 1.0 μl of a multiplex primer set (1 pM of each primer) prepared for hantaviral multiplex PCR, 12 μl of 2 × Uh-buffer, 0.8 μl of Uh-Taq (2.5 units) A 25 μl reaction mixture was prepared by mixing the molecular-grade water and then subjected to a first PCR in a ABI veriti 96-well gene cycler (Thermal Cycler). The first PCR reaction conditions were initial denaturation at 95 캜 for 15 minutes, followed by denaturation at 95 캜 for 20 seconds, annealing at 50 캜 for 40 seconds, and extension at 72 캜 for 1 minute. Cycle (amplification step) was performed and an additional extension (final extension step) at 72 ° C for 3 minutes (Table 7). The nucleic acid amplification product amplified by the first PCR was electrophoresed on 3% agarose gel (Cat 11 685 678 001, Roche, Germany). As a result, amplification of the target detection gene was confirmed based on the standard marker.

Primary PCR reaction liquid composition and reaction conditions. PCR  Reaction liquid composition PCR  Reaction conditions Temperature time cycle Template
( cDNA diluent ) 3.0 μl 95 ℃ 15 minutes One
(Denaturation step) primer  mixture
(Each 1 pM ) 1.0 μl 95 ℃ 20 seconds 40
(Amplification step) 2x Uh - Buffer 12.5 μl 50 ℃ 40 seconds Uh - Taq
(2.5 Unit ) 0.8 μl 72 ℃ 1 minute water 7.7 μl 72 ℃ 3 minutes One
(Final extension step) synthesis 25.0 μl 10 ℃ Keep

FIG. 3 shows the result of the experiment using the multiplex primer set pool of FIG. 1 produced by the present invention. In multiplexer primer set 1 and set 2 of Hanta virus L, M or S segments in two isolate strains, (1- st PCR product). The appearance of the reaction product on the agarose gel was confirmed in the form of a ladder as illustrated in Fig.

FIG. 4 is a PCR experiment using the multiplex primer set of FIG. 1 designed and manufactured by the method of the present invention. In multiplex type primer set 1 of hanta virus L, M or S segments in five kinds of liver tissues, (1 st PCR product). The results are shown in Fig.

2-4. Multiplex secondary PCR  Reaction and confirmation

1.0 μl of the multiplex PCR primer set (1 pM of each primer), 12.5 μl of 2 × Uh buffer, and 2.5 μl of Uh-Taq (2.5 μl each) using 1.0 μl of the first PCR product performed using the multiplex primer set as a template Unit), and 25 μl of the reaction mixture were mixed with the molecular-grade water, followed by secondary PCR in ABI veriti 96-well Thermal Cycler. The secondary PCR reaction conditions were initial denaturation at 95 캜 for 15 minutes followed by denaturation at 95 캜 for 20 seconds, annealing at 50 캜 for 40 seconds, and extension at 72 캜 for 1 minute Cycle (amplification step) was performed and an additional extension (final extension step) at 72 ° C for 3 minutes (Table 8). The nucleic acid amplification product amplified by the secondary PCR was electrophoresed on 3% agarose gel. As a result, amplification of the target detection gene was confirmed based on the standard marker.

Second PCR reaction liquid composition and reaction conditions. PCR  Reaction liquid composition PCR  Reaction conditions Temperature time cycle Template
(Primary PCR  product) 1.0 μl 95 ℃ 15 minutes One
(Denaturation step) primer  mixture
(Each 1 pM ) 1.0 μl 95 ℃ 20 seconds 40
(Amplification step) 2x Uh - Buffer 12.5 μl 50 ℃ 40 seconds Uh - Taq
(2.5 Unit ) 0.8 μl 72 ℃ 1 minute water 9.7 μl 72 ℃ 3 minutes One
(Final extension step) synthesis 25.0 μl 10 ℃ Keep

FIG. 5 shows the result of the experiment using the multiplex primer of FIG. 1 designed and manufactured in the present invention. In the 15 patient samples, primer set 1 for Hantavirus L, M and S segments was used to amplify polymerase The result of the chain reaction was diluted to an appropriate amount, and further secondary PCR was performed (2 nd PCR product).

2-5. Sample preparation and purification for Hantaa virus full-length genome sequencing NGS  Perform

Multiplex secondary PCR products are fragmented using a Covaris Focused-Ultrasonicator. The fragmented PCR sample was cleaned up using magnetic beads (Illumina, USA) and the end repair reaction was performed using 40 μl of the end repari mix (Illumina, USA) Lt; 0 > C for 30 minutes. The magnetic beads then removed all unwanted large size and small DNA fragments. Size The selected PCR samples were reacted with 12.5 μl of A-tailing mix (Illumina, USA) for 30 minutes at 37 ° C, 5 minutes at 70 ° C and 5 minutes at 4 ° C. The PCR sample thus treated was subjected to a ligation reaction at 30 ° C for 10 minutes using 2.5 μl of an illuminating buffer, 2.5 μl of a ligation mixture (Illumina, USA), and 2.5 μl of a specific DNA adapter. After 10 minutes, 5 μl of stop solution was added to stop the reaction. For the samples that had undergone two purification steps, 5 μl of PCR primer cocktail and 20 μl of the enhanced PCR mixture were added, and the amount of the sample was amplified through a polymerase chain reaction. The reaction conditions include 8 cycles (denaturation at 95 DEG C for 3 minutes, denaturation at 98 DEG C for 20 seconds, annealing at 60 DEG C for 15 seconds, and extension at 72 DEG C for 30 seconds Amplification step) was performed and further extension (final extension step) for 5 minutes at 72 ° C. After this sort of reaction, it is subjected to one purification process and prepared for running on the NGS equipment through quality control. After confirming the concentration and quality of the sample through bio-analyzer and RT-qPCR, NGS reaction was performed using Illumina MiSeq equipment.

Figure 6 shows the results of comparing consensus PCR with SISPA-NGS to compare the accuracy and homology of NGS results based on multiplex, and compared with SISPA-NGS on the basis of consensus PCR, The multiplex PCR-based NGS developed by the present invention has high homology.

FIG. 7 is a graph showing the results of the SISPA (Sequence Independent, Single Primer Amplification; Molecular and Cellular Probes, Volume 5, Issue 6, December 1991, Pages 473-481) method, which is generally used for obtaining the full-length genome sequence (WGS) And the FASTQ file, which is a result of the reaction with MiSeq, an NGS instrument, is mapped to L, M, and S segment sequences using a reference genome sequence of Hanta virus. As can be seen in FIG. 7, the coverage at the distal end of each segment was low, and it was found that a gap was generated in the L segment having a longer length than the other segments.

FIG. 8 is a graph showing the results of a pre-treatment of a sample using a multiplex primer set designed and fabricated according to the present invention, and a FASTQ file as a result of the reaction with MiSeq, an NGS instrument, And S segmentation sequences, respectively. As can be seen from FIG. 8, the overall distribution of each segment was high and uniform, and no gap occurred at all, indicating that the full-length genome sequence of the virus can be obtained at one time.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Agency for Defense Development <120> Hantavirus Whole Genome Sequence Identification Methods Using a          Multiplex PCR-based Next-Generation Sequencing and Uses Thereof <130> PT20150135 <160> 473 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL01F <400> 1 tagtagtaga ctccggaagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL02F <400> 2 gtagagtgta tagattatct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL03F <400> 3 aggcaatagg gaaagtacta 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL04F <400> 4 caaggccttt ttcaagatga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL05F <400> 5 agagaaaaga agcttaaata 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL06F <400> 6 catttaatgt agtggcagtt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL07F <400> 7 agctgagata agttacgtta 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL08F <400> 8 agccagccat attacatacc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL09F <400> 9 gaaatttttc attttttgaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL10F <400> 10 tagaaacttc ttactaatac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL11F <400> 11 ctgaacttaa ttccagatac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL12F <400> 12 cacgaataca atttaaacag 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL13F <400> 13 actcaataat cataaaagtg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL14F <400> 14 aaaatattgt ctgatttaga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL15F <400> 15 ttcagaatga gacccagaag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL16F <400> 16 tcattcagga ttaaaaagat 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL17F <400> 17 ggttccttta ttaggttcat 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL18F <400> 18 gggtatccaa agtcatgagc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL19F <400> 19 tacagaagac caagggcagt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL20F <400> 20 atttttgaca atctacgtta 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL21F <400> 21 cattagaggt atatatatat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL22F <400> 22 aacagtggac cagtcgactg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL23F <400> 23 acctgtttct ttttatttga 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL24F <400> 24 aggaagaaaa gtatggggaa 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL25F <400> 25 tgctgttgag ttggcagcaa 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL26F <400> 26 tttagccaaa caagaaatat 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL27F <400> 27 atttatcaaa ttcaagacat 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL28F <400> 28 agaggcagat aggggctttt 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL29F <400> 29 gagtacatat catatggagg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL30F <400> 30 gtaatcataa attcattagg 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL31F <400> 31 tacctccatg ctacataatg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL32F <400> 32 aaactaagga actatattga 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL33F <400> 33 actggctgca gggtaactta 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL34F <400> 34 ttgctttttt gagtttgcac 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL35F <400> 35 caacagattc aagcaggcca 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL36F <400> 36 ttaagatctc accaaagaaa 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL37F <400> 37 actcttaggt tctctgtcag 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL38F <400> 38 cctcaggttg cacaactagc 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL39F <400> 39 aagttaagca tgctgataca 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL40F <400> 40 attaaagcgc gcacttctag 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL41F <400> 41 tttcaacatg agaggttagg 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL42F <400> 42 caaaattttt agagttatgg 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL43F <400> 43 tgacccaagt attgttacag 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL44F <400> 44 gaatgggtta ccttcaggga 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL45F <400> 45 gttgtacatt ttctaaagag 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL46F <400> 46 aacattcaca gtcagagaaa 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL47F <400> 47 ctggatacag ccaagttccc 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL48F <400> 48 ttatgaaaag aattgcacct 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL49F <400> 49 ctggctgaaa tcaatgtcac 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL50F <400> 50 caacagaaag acttagctgc 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL51F <400> 51 tcgaggataa gaccttttca 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL52F <400> 52 tataaagggt gattattcag 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL53F <400> 53 acaaagcttt atgaaggaga 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL54F <400> 54 catcaattac acagaagcat 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL55F <400> 55 agtacggcac atggtattgt 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL56F <400> 56 cttagaacca cacaggcaat 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL57F <400> 57 tattagctca tgcgtttcat 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL58F <400> 58 aaatcccatc atttcatctg 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL59F <400> 59 atatctgttg atctctttat 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL60F <400> 60 gtactgtgag ttcaatgtca 20 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL61F <400> 61 tgtggatttt gagaatatag 20 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL62F <400> 62 gaatcaaaga aaatgagggg 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL63F <400> 63 ctgttaatat tatcttaatg 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL64F <400> 64 gagcattgtc cgaggatggg 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL65F <400> 65 gatgtcattc ctgattcatt 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL01R <400> 66 atgtcatgtc tcacggcgta 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL02R <400> 67 tataatgttt gaagggaccc 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL03R <400> 68 ttgttccatt aattttataa 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL04R <400> 69 tcttgttcaa tataagctaa 20 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL05R <400> 70 ttgtgtagta atattggagc 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL06R <400> 71 ctctctcctc agttctgatc 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL07R <400> 72 gctcctatcg gttccatccc 20 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL08R <400> 73 aaagcactcg aacactgctg 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL09R <400> 74 caggtttgta agatgtgatt 20 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL10R <400> 75 gccatgtcat ggatcaggta 20 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL11R <400> 76 aaatgttcct ggctctctta 20 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL12R <400> 77 gactctctat ttgcgcacca 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL13R <400> 78 tcttgagttg atggctcagt 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL14R <400> 79 tgttgtcttt ttcaacacat 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL15R <400> 80 tattgtatga gtgtaaggac 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL16R <400> 81 actaaaccag gtcctatcct 20 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL17R <400> 82 atttaatgct agcagcctat 20 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL18R <400> 83 acacagatct aattgcatat 20 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL19R <400> 84 gagtataatg atgtcactgc 20 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL20R <400> 85 aagtgcaact aacaagcttt 20 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL21R <400> 86 taaatgaagg gtaaactcca 20 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL22R <400> 87 tcttcattca tatttccatg 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL23R <400> 88 ccacatcata tatccatttt 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL24R <400> 89 actttgtagc caggacttta 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL25R <400> 90 tgaacctgtc ctgacatacc 20 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL26R <400> 91 ctcctcatat aattttaaga 20 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL27R <400> 92 gcctgcatct agtaggaagt 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL28R <400> 93 gccccttgga ttgcaagaat 20 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL29R <400> 94 tgcactaaca tacatgagtt 20 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL30R <400> 95 cacagttctt tagcttattg 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL31R <400> 96 ttgatgtgtg ggtcaaggct 20 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL32R <400> 97 tgcaacaccg aaaagcgaag 20 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL33R <400> 98 cataaatgaa taatgcatcg 20 <210> 99 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL34R <400> 99 atctctgtat ttacactaaa 20 <210> 100 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL35R <400> 100 aaattcagca tttgtcgggg 20 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL36R <400> 101 aatcgtcaaa ataacctaaa 20 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL37R <400> 102 ctttcaactt tactggtgca 20 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL38R <400> 103 cattgcacca tttcctccca 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL39R <400> 104 gcattgattt ctgtctcttg 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL40R <400> 105 cactgtactt tcccaataaa 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL41R <400> 106 aatataatca taagttacat 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL42R <400> 107 ggagctgcaa gggtgactgc 20 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL43R <400> 108 tctgcaaaac tgttagcagc 20 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL44R <400> 109 aattccattc aagaaatctt 20 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL45R <400> 110 caggaatact attttgtatg 20 <210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL46R <400> 111 attgtcttta agtcaacaga 20 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL47R <400> 112 tacccttgat ttagaagatt 20 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL48R <400> 113 tatgaggctt cactcttata 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL49R <400> 114 catctccaaa tttctatgca 20 <210> 115 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL50R <400> 115 cccaacccta cagaaccgat 20 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL51R <400> 116 atgctcgcct gttggcttgt 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL52R <400> 117 tcaagaccaa aattgaatgt 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL53R <400> 118 cgaaacctca tccatcatga 20 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL54R <400> 119 tcccccattc aaatccagac 20 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL55R <400> 120 tcctttattg taatcataaa 20 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL56R <400> 121 tactgtttga tacctaatat 20 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL57R <400> 122 gaagattctt cataaaattc 20 <210> 123 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL58R <400> 123 gggttaatca gcttaagtaa 20 <210> 124 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL59R <400> 124 taagctcatt tcttcagacc 20 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL60R <400> 125 gtaggaaatg ttcaatatct 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL61R <400> 126 agtctgacag gctcaagtat 20 <210> 127 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL62R <400> 127 caaattaaat gttttagaga 20 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL63R <400> 128 gattttcaaa ctgatcaata 20 <210> 129 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL64R <400> 129 aacagtacca tggtatgcct 20 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNL65R <400> 130 ttagtagtag gtatgctccg 20 <210> 131 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L01F <400> 131 accctgaaaa gaatggataa 20 <210> 132 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L02F <400> 132 gactatacgc cgtgagacat 20 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L03F <400> 133 tgcaggggtc ccttcaaaca 20 <210> 134 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L04F <400> 134 gataattata aaattaatgg 20 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L05F <400> 135 caggcttagc ttatattgaa 20 <210> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L06F <400> 136 agatggctcc aatattacta 20 <210> 137 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L07F <400> 137 cacttgatca gaactgagga 20 <210> 138 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L08F <400> 138 ataaggggat ggaaccgata 20 <210> 139 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L09F <400> 139 aggtacagca gtgttcgagt 20 <210> 140 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L10F <400> 140 tctttaatca catcttacaa 20 <210> 141 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L11F <400> 141 catcttacct gatccatgac 20 <210> 142 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L12F <400> 142 gaatataaga gagccaggaa 20 <210> 143 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L13F <400> 143 aagcatggtg cgcaaataga 20 <210> 144 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L14F <400> 144 atataactga gccatcaact 20 <210> 145 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L15F <400> 145 gatagatgtg ttgaaaaaga 20 <210> 146 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L16F <400> 146 tattggtcct tacactcata 20 <210> 147 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L17F <400> 147 tttttaggat aggacctggt 20 <210> 148 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L18F <400> 148 tttaaatagg ctgctagcat 20 <210> 149 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L19F <400> 149 ttacaatatg caattagatc 20 <210> 150 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L20F <400> 150 tacctgcagt gacatcatta 20 <210> 151 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L21F <400> 151 tattaaaagc ttgttagttg 20 <210> 152 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L22F <400> 152 gcaagtggag tttacccttc 20 <210> 153 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L23F <400> 153 gtctccatgg aaatatgaat 20 <210> 154 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L24F <400> 154 agtagaaaat ggatatatga 20 <210> 155 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L25F <400> 155 ttgaataaag tcctggctac 20 <210> 156 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L26F <400> 156 taaaaggtat gtcaggacag 20 <210> 157 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L27F <400> 157 tagcctctta aaattatatg 20 <210> 158 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L28F <400> 158 acaacacttc ctactagatg 20 <210> 159 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L29F <400> 159 aaaagattct tgcaatccaa 20 <210> 160 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L30F <400> 160 acgtaaactc atgtatgtta 20 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L31F <400> 161 cctaacaata agctaaagaa 20 <210> 162 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L32F <400> 162 tggaaagcct tgacccacac 20 <210> 163 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L33F <400> 163 gtgttcttcg cttttcggtg 20 <210> 164 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L34F <400> 164 tctgacgatg cattattcat 20 <210> 165 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L35F <400> 165 actggtttag tgtaaataca 20 <210> 166 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L36F <400> 166 agtgtccccg acaaatgctg 20 <210> 167 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L37F <400> 167 ccaggtttag gttattttga 20 <210> 168 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L38F <400> 168 cattgtgcac cagtaaagtt 20 <210> 169 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L39F <400> 169 tccattggga ggaaatggtg 20 <210> 170 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L40F <400> 170 tcacacaaga gacagaaatc 20 <210> 171 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L41F <400> 171 tttcttttat tgggaaagta 20 <210> 172 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L42F <400> 172 tcaacatgta acttatgatt 20 <210> 173 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L43F <400> 173 accatgcagt cacccttgca 20 <210> 174 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L44F <400> 174 tggctgctgc taacagtttt 20 <210> 175 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L45F <400> 175 ttggaaagat ttcttgaatg 20 <210> 176 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L46F <400> 176 cagatcatac aaaatagtat 20 <210> 177 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L47F <400> 177 ctctatctgt tgacttaaag 20 <210> 178 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L48F <400> 178 ctataaatct tctaaatcaa 20 <210> 179 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L49F <400> 179 aagactataa gagtgaagcc 20 <210> 180 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L50F <400> 180 agttgtgcat agaaatttgg 20 <210> 181 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L51F <400> 181 gttagatcgg ttctgtaggg 20 <210> 182 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L52F <400> 182 cagttacaag ccaacaggcg 20 <210> 183 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L53F <400> 183 gagtaacatt caattttggt 20 <210> 184 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L54F <400> 184 aaagatcatg atggatgagg 20 <210> 185 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L55F <400> 185 acagagtctg gatttgaatg 20 <210> 186 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L56F <400> 186 ataaatttat gattacaata 20 <210> 187 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L57F <400> 187 aagagatatt aggtatcaaa 20 <210> 188 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L58F <400> 188 cttgagaatt ttatgaagaa 20 <210> 189 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L59F <400> 189 atgatttact taagctgatt 20 <210> 190 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L60F <400> 190 attatggtct gaagaaatga 20 <210> 191 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L61F <400> 191 gaagcagata ttgaacattt 20 <210> 192 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L62F <400> 192 tgaaaatact tgagcctgtc 20 <210> 193 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L63F <400> 193 atatatctct aaaacattta 20 <210> 194 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L64F <400> 194 tgtgttattg atcagtttga 20 <210> 195 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L65F <400> 195 catttaggca taccatggta 20 <210> 196 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L01R <400> 196 gtcatgctta atcatttggt 20 <210> 197 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L02R <400> 197 gtattatctt tttttctagt 20 <210> 198 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L03R <400> 198 acagtaacct caacaaactc 20 <210> 199 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L04R <400> 199 acccttcaga aagaacttat 20 <210> 200 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L05R <400> 200 caccatcatt tcttctactg 20 <210> 201 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L06R <400> 201 agattaaaca tggcttctag 20 <210> 202 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L07R <400> 202 atagtccact aaatcttcaa 20 <210> 203 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L08R <400> 203 gacaatgatt ggcctcattt 20 <210> 204 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L09R <400> 204 tcaatttgat ctgacaatgt 20 <210> 205 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L10R <400> 205 tcttgttagg tttatgtatc 20 <210> 206 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L11R <400> 206 ctctcagcat acttgatgtc 20 <210> 207 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L12R <400> 207 tgtacaatat gactagcaat 20 <210> 208 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L13R <400> 208 ataagtatgc ttagttgtgg 20 <210> 209 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L14R <400> 209 ttatgagatg acctatgtgc 20 <210> 210 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L15R <400> 210 agaataaaca atatgacatt 20 <210> 211 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L16R <400> 211 tatagtatcc aaattatctt 20 <210> 212 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L17R <400> 212 caattaaagc cttttcgaaa 20 <210> 213 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L18R <400> 213 atagctaaca aaaaatgatt 20 <210> 214 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L19R <400> 214 tttttctatc agtgaaggaa 20 <210> 215 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L20R <400> 215 agaatctagc tttattattt 20 <210> 216 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L21R <400> 216 taatgtttgt atactatacg 20 <210> 217 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L22R <400> 217 aactgtctca agatggattt 20 <210> 218 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L23R <400> 218 ctaactcagc attctcccgt 20 <210> 219 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L24R <400> 219 aatatgctat tagcgacaac 20 <210> 220 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L25R <400> 220 agatgatgat aagtggccat 20 <210> 221 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L26R <400> 221 ttgccattgc tttctgctct 20 <210> 222 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L27R <400> 222 tcattgtagt cctcaataat 20 <210> 223 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L28R <400> 223 tgcccatctc aaggccttct 20 <210> 224 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L29R <400> 224 cccctggtga ccactttgta 20 <210> 225 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L30R <400> 225 taaacttgtt taagtgcatc 20 <210> 226 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L31R <400> 226 atcagggaaa aaatccagaa 20 <210> 227 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L32R <400> 227 atacctgttt aaataataat 20 <210> 228 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L33R <400> 228 ccatcatcta caggttccaa 20 <210> 229 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L34R <400> 229 atgtagatta aacatactct 20 <210> 230 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L35R <400> 230 cacaaccctc aaaaaatgtt 20 <210> 231 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L36R <400> 231 acacatctgc tttgtgctgc 20 <210> 232 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L37R <400> 232 catacctggt gctgtaccat 20 <210> 233 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L38R <400> 233 cagctgttgc taactccatt 20 <210> 234 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L39R <400> 234 aacttgaata gacctaatat 20 <210> 235 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L40R <400> 235 ttcagatttt ggagtgaaga 20 <210> 236 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L41R <400> 236 gaagattgtc tctgcccttt 20 <210> 237 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L42R <400> 237 tgctgcttgg cttgcattct 20 <210> 238 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L43R <400> 238 atcttggcta gtagcactat 20 <210> 239 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L44R <400> 239 gttttgttgg gataacatca 20 <210> 240 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L45R <400> 240 acagcaaatt tgtacccaat 20 <210> 241 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L46R <400> 241 cagctcacga taaactccat 20 <210> 242 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L47R <400> 242 cttcaacatt tccttggaca 20 <210> 243 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L48R <400> 243 attgaaactg cttgaaggac 20 <210> 244 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L49R <400> 244 atatggagca ctatttgctt 20 <210> 245 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L50R <400> 245 ggatctctgg atcaattggt 20 <210> 246 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L51R <400> 246 tatgcatcat attgcttacc 20 <210> 247 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L52R <400> 247 ccaaaagatc tcaagccttg 20 <210> 248 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L53R <400> 248 tcatcccaca cttaattagc 20 <210> 249 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L54R <400> 249 taaacaatgc atggaatatt 20 <210> 250 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L55R <400> 250 ttgtgcaata gcacgaagcc 20 <210> 251 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L56R <400> 251 tgaaccatac attgcttaca 20 <210> 252 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L57R <400> 252 taagcagcag gagggattgc 20 <210> 253 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L58R <400> 253 gcctgataga tccaatgtat 20 <210> 254 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L59R <400> 254 catcaagctc ttcatcatca 20 <210> 255 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L60R <400> 255 tctcctgtgt atgaactctc 20 <210> 256 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L61R <400> 256 tccacgtgat acccagcttt 20 <210> 257 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L62R <400> 257 ccaataatga gacctgtttg 20 <210> 258 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L63R <400> 258 accatacttt gagcctctct 20 <210> 259 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L64R <400> 259 agaatcctgt ggaaataacc 20 <210> 260 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2L65R <400> 260 ttagtagtag gtatgctccg 20 <210> 261 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> HNM01F <400> 261 tagtagtaga ctccgcaaaa taaag 25 <210> 262 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNMO2F <400> 262 aatgtctatg acatgaaaat 20 <210> 263 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNMO3F <400> 263 cagcacagct ggtgcctgag 20 <210> 264 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM04F <400> 264 tcagtcacag tctagccaaa 20 <210> 265 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM05F <400> 265 cgcagtagaa aatcaataac 20 <210> 266 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM06F <400> 266 tgaaaagctg tttgattgca 20 <210> 267 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM07F <400> 267 cccagatcag agtgtggtca 20 <210> 268 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM08F <400> 268 aaaatttttg aacaggttaa 20 <210> 269 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM09F <400> 269 caccaattta tgttccaaca 20 <210> 270 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM10F <400> 270 agaaactgca acttactcta 20 <210> 271 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM11F <400> 271 tcttattctt cccttagtat 20 <210> 272 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM12F <400> 272 gtgccatacc actcatgtgg 20 <210> 273 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM13F <400> 273 atcatgtgag gccttttctg 20 <210> 274 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM14F <400> 274 aattttgtat gtcagcgagt 20 <210> 275 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM15F <400> 275 acactataac aagcttattt 20 <210> 276 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM16F <400> 276 gcttgttaca ttctgttttg 20 <210> 277 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM17F <400> 277 caagaaaaca ggttaaaatc 20 <210> 278 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM18F <400> 278 cctataaaga gttgaaggca 20 <210> 279 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM19F <400> 279 ttacaaagta tgccaagtta 20 <210> 280 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM20F <400> 280 tttagataca aaagtaggtg 20 <210> 281 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM21F <400> 281 ctcctgtctg gaatgacaat 20 <210> 282 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM22F <400> 282 ccgtaggaag ctaacaaacc 20 <210> 283 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM23F <400> 283 cactggtttg atggtcgtct 20 <210> 284 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM24F <400> 284 aaagggatta ccaatatgag 20 <210> 285 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM25F <400> 285 gaaaccagtt ggcagtgctt 20 <210> 286 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM26F <400> 286 aatgactgtt ttgtatctag 20 <210> 287 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM27F <400> 287 gtggcggtct aatatttaaa 20 <210> 288 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM28F <400> 288 gttcccaggt agtttcagga 20 <210> 289 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM29F <400> 289 attgattcct ttcaatcttt 20 <210> 290 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM30F <400> 290 tcttagtaac aaaagacatc 20 <210> 291 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM31F <400> 291 agggtttaca ttaacatgtc 20 <210> 292 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM32F <400> 292 gtaacattaa caagaggaca 20 <210> 293 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM33F <400> 293 ttggactcca tgctgctgca 20 <210> 294 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM34F <400> 294 aaaatgttgg tttgttaaat 20 <210> 295 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM35F <400> 295 gttttactga gcattctctg 20 <210> 296 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM36F <400> 296 tttttatatt ccagtataat 20 <210> 297 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM01R <400> 297 tccccgaagc ttactgtgtg 20 <210> 298 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM02R <400> 298 tgattggtga ttatccatgc 20 <210> 299 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM03R <400> 299 caacttcagt agacactgtc 20 <210> 300 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM04R <400> 300 taagtactat tgcatgatag 20 <210> 301 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM05R <400> 301 ataaactacc tgtactctgt 20 <210> 302 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM06R <400> 302 cgatatcaaa gatcccatgc 20 <210> 303 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM07R <400> 303 gtatcattgc atgttgattc 20 <210> 304 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM08R <400> 304 gaatgcttcc atggatctga 20 <210> 305 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM09R <400> 305 gaggaacttt tgcattggca 20 <210> 306 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM10R <400> 306 acatgtttgg cttctgttga 20 <210> 307 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM11R <400> 307 ataatatcca ggtaaatcaa 20 <210> 308 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM12R <400> 308 tgggggaggt tatgttgaaa 20 <210> 309 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM13R <400> 309 ccattgcagt acacaacgat 20 <210> 310 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM14R <400> 310 aatagagtgt gctaccccag 20 <210> 311 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM15R <400> 311 taaatgtgat tgctggtata 20 <210> 312 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM16R <400> 312 tcaaactctt cctttatctt 20 <210> 313 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM17R <400> 313 gcattgagac tgggggcatg 20 <210> 314 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM18R <400> 314 ttttctttag atcatccctg 20 <210> 315 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM19R <400> 315 agaagaaata tccacattgt 20 <210> 316 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM20R <400> 316 gtgcatagga acagaaccca 20 <210> 317 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM21R <400> 317 gtagatcaat ggattgtgct 20 <210> 318 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM22R <400> 318 ccataacagt gaaaggatgt 20 <210> 319 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM23R <400> 319 acaatctgat ggattacaac 20 <210> 320 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM24R <400> 320 tcctgctata ccttattgtg 20 <210> 321 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM25R <400> 321 attgtaccaa ttatacagac 20 <210> 322 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM26R <400> 322 gccaaattga catgtggatg 20 <210> 323 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM27R <400> 323 aaataggagt agtagcaaag 20 <210> 324 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM28R <400> 324 tcatcagtaa agtgcatagt 20 <210> 325 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM29R <400> 325 gcaaggattt tcaccaaggt 20 <210> 326 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM30R <400> 326 aaaaggtagg acattctgtt 20 <210> 327 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM31R <400> 327 ccaccttttc ctgacacctt 20 <210> 328 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM32R <400> 328 agaaatccca ttgaccttgt 20 <210> 329 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM33R <400> 329 cactgaatat ccctgaaatc 20 <210> 330 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM34R <400> 330 agctatgact ttttatgctt 20 <210> 331 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNM35R <400> 331 gctactattt ttttttagtg 20 <210> 332 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> HNM36R <400> 332 tagtagtaga ctccgcaaaa tgtta 25 <210> 333 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M01F <400> 333 cagtcaatca acaacatggg 20 <210> 334 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M02F <400> 334 gcccccacac agtaagcttc 20 <210> 335 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M03F <400> 335 ttgcagcatg gataatcacc 20 <210> 336 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M04F <400> 336 tttgagacag tgtctactga 20 <210> 337 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M05F <400> 337 atgacctatc atgcaatagt 20 <210> 338 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M06F <400> 338 accatacaga gtacaggtag 20 <210> 339 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M07F <400> 339 atcaagcatg ggatctttga 20 <210> 340 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M08F <400> 340 cctttgaatc aacatgcaat 20 <210> 341 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M09F <400> 341 tgatttcaga tccatggaag 20 <210> 342 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M10F <400> 342 ggacctgcca atgcaaaagt 20 <210> 343 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M11F <400> 343 caagttcaac agaagccaaa 20 <210> 344 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M12F <400> 344 gatgattgat ttacctggat 20 <210> 345 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M13F <400> 345 gggattttca acataacctc 20 <210> 346 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M14F <400> 346 tggacatcgt tgtgtactgc 20 <210> 347 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M15F <400> 347 actgcctggg gtagcacact 20 <210> 348 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M16F <400> 348 gttcttatac cagcaatcac 20 <210> 349 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M17F <400> 349 ttagaaagat aaaggaagag 20 <210> 350 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M18F <400> 350 tgtgtcatgc ccccagtctc 20 <210> 351 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M19F <400> 351 aggttcaggg atgatctaaa 20 <210> 352 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M20F <400> 352 tctttacaat gtggatattt 20 <210> 353 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M21F <400> 353 tggagtgggt tctgttccta 20 <210> 354 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M22F <400> 354 gaagaagcac aatccattga 20 <210> 355 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M23F <400> 355 ttaaaacatc ctttcactgt 20 <210> 356 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M24F <400> 356 ctggggttgt aatccatcag 20 <210> 357 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M25F <400> 357 atcatcacaa taaggtatag 20 <210> 358 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M26F <400> 358 ttaaggtctg tataattggt 20 <210> 359 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M27F <400> 359 gtgcacatcc acatgtcaat 20 <210> 360 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M28F <400> 360 tgcaactttg ctactactcc 20 <210> 361 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M29F <400> 361 caagtactat gcactttact 20 <210> 362 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M30F <400> 362 tgataacctt ggtgaaaatc 20 <210> 363 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M31F <400> 363 tcgctaacag aatgtcctac 20 <210> 364 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M32F <400> 364 cagtcaaggt gtcaggaaaa 20 <210> 365 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M33F <400> 365 cctagacaag gtcaatggga 20 <210> 366 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M34F <400> 366 gaatggattt cagggatatt 20 <210> 367 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M35F <400> 367 tgaggaagca taaaaagtca 20 <210> 368 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M36F <400> 368 taacacacta aaaaaaaata 20 <210> 369 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M01R <400> 369 ttccacatag cctatcacac 20 <210> 370 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M02R <400> 370 gggtatattt tgttattgta 20 <210> 371 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M03R <400> 371 ttcagaacac atgttccttt 20 <210> 372 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M04R <400> 372 aatcatgtat agtgttggct 20 <210> 373 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M05R <400> 373 ctcctgtcat gcaataggtt 20 <210> 374 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M06R <400> 374 aagaagcaca caatatgaac 20 <210> 375 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M07R <400> 375 ataatacccc tgcactttat 20 <210> 376 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M08R <400> 376 catgtggtga tctgaagatt 20 <210> 377 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M09R <400> 377 ctcaatgtat ctgagctagc 20 <210> 378 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M10R <400> 378 ttttgggaac aacccagggc 20 <210> 379 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M11R <400> 379 atactgtaca ggggtggata 20 <210> 380 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M12R <400> 380 cgattttgtt ttgacactaa 20 <210> 381 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M13R <400> 381 ttttgtcaat attacttttc 20 <210> 382 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M14R <400> 382 atccaggtac acacaattca 20 <210> 383 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M15R <400> 383 atgaacttta ggattgtcag 20 <210> 384 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M16R <400> 384 acataccatc gagccttttg 20 <210> 385 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M17R <400> 385 gttcacaatg agtaaagcaa 20 <210> 386 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M18R <400> 386 ggtgtgaagt tctgaggagt 20 <210> 387 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M19R <400> 387 agcccatagt atagattcta 20 <210> 388 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M20R <400> 388 aagagaaatc aagttctaaa 20 <210> 389 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M21R <400> 389 attgtttgtt cttctatttc 20 <210> 390 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M22R <400> 390 agggtattca tattttgtac 20 <210> 391 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M23R <400> 391 ctgtacagcc tgtgcccacc 20 <210> 392 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M24R <400> 392 tcttccccga attgaacaca 20 <210> 393 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M25R <400> 393 tgtatcaccc tgagagaatt 20 <210> 394 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M26R <400> 394 ttgggctcat aatgtctcct 20 <210> 395 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M27R <400> 395 gagaccatat ttccgtcata 20 <210> 396 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M28R <400> 396 atcagggtct ttccactcta 20 <210> 397 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M29R <400> 397 tggaagatgc ctgtaggcca 20 <210> 398 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M30R <400> 398 ttatcacaag cctttattga 20 <210> 399 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M31R <400> 399 acacttaaat gttgaaccac 20 <210> 400 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M32R <400> 400 catatacttt gctattttcc 20 <210> 401 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M33R <400> 401 agaacaatga gtacaatcca 20 <210> 402 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M34R <400> 402 caataacagg atagtgacag 20 <210> 403 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2M35R <400> 403 agaatctaag ttttgtggtt 20 <210> 404 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> HN2M36R <400> 404 tagtagtaga ctccgcaaaa tgtta 25 <210> 405 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS01F <400> 405 tagtagtagg ctccctaaag 20 <210> 406 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS02F <400> 406 ggcagaaggt aagggatgca 20 <210> 407 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS03F <400> 407 ggcaaagatt gatgagttaa 20 <210> 408 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS04F <400> 408 catctgaaag agagatcaat 20 <210> 409 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS05F <400> 409 ttgttgtcta tcttacatcc 20 <210> 410 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS06F <400> 410 tcggattcga tttaaggatg 20 <210> 411 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS07F <400> 411 aaggcagaag agattacacc 20 <210> 412 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS08F &Lt; 400 > 412 tgattggttt cctggcattg 20 <210> 413 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS09F <400> 413 ccttggtggt cctgcaacaa 20 <210> 414 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS10F <400> 414 gaagcagctg gctgtagcat 20 <210> 415 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS11F <400> 415 tagcaggtat tgctgagctt 20 <210> 416 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS12F <400> 416 gcgaaagaaa tcatcattct 20 <210> 417 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS13F <400> 417 ggaaaggagg ctgtggacaa 20 <210> 418 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS14F <400> 418 ccaaccaaga gcctttaaaa 20 <210> 419 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS15F <400> 419 ttatcagggg aatcagtata 20 <210> 420 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS16F <400> 420 aggctgcctt aagtagcctt 20 <210> 421 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS17F <400> 421 tactaacaac aacactctac 20 <210> 422 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS01R <400> 422 ctcatctgga tcctttccat 20 <210> 423 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS02R <400> 423 cagttgcaat cctatctgcc 20 <210> 424 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS03R <400> 424 tttaaatcca gcacattacc 20 <210> 425 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS04R <400> 425 tagagctttc agaagtatcg 20 <210> 426 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS05R <400> 426 ggataccgtt aacgtcctcg 20 <210> 427 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS06R <400> 427 agtccacaga ctgctgttct 20 <210> 428 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS07R <400> 428 ttgttcaata cgatcactcc 20 <210> 429 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS08R <400> 429 ccacttgccg ctgccgtaag 20 <210> 430 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS09R <400> 430 attgatgatg gtgactcaat 20 <210> 431 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS10R <400> 431 catgtcctgc aggatggaaa 20 <210> 432 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS11R <400> 432 ttgattgtgt ccttctgaga 20 <210> 433 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS12R <400> 433 tcaggatcca tatcatcccc 20 <210> 434 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS13R <400> 434 taaaaggatt aatacattca 20 <210> 435 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS14R <400> 435 ttcccaccca taaatgttcc 20 <210> 436 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS15R <400> 436 aaatgaaatc tacatccata 20 <210> 437 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS16R <400> 437 gtagttaagt tgaggtagtt 20 <210> 438 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNS17R <400> 438 gtagtagttt tgctccctaa 20 <210> 439 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S01F <400> 439 ctagaacaac gatggcaact 20 <210> 440 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S02F &Lt; 400 > 440 acagtatgga aaggatccag 20 <210> 441 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S03F <400> 441 cagctggcag ataggattgc 20 <210> 442 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S04F <400> 442 gttatggtaa tgtgctggat 20 <210> 443 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S05F <400> 443 tgtcccgata cttctgaaag 20 <210> 444 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S06F <400> 444 tcgttcgagg acgttaacgg 20 <210> 445 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S07F <400> 445 gatatagaac agcagtctgt 20 <210> 446 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S08F <400> 446 ggattggagt gatcgtattg 20 <210> 447 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S09F <400> 447 gactacttac ggcagcggca 20 <210> 448 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S10F <400> 448 aggacattga gtcaccatca 20 <210> 449 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S11F <400> 449 atttttttcc atcctgcagg 20 <210> 450 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S12F <400> 450 tcttatctca gaaggacaca 20 <210> 451 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S13F <400> 451 acttagggga tgatatggat 20 <210> 452 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S14F <400> 452 atcaatgaat gtattaatcc 20 <210> 453 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S15F <400> 453 gcaagggaac atttatgggt 20 <210> 454 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S16F <400> 454 gtatatatgg atgtagattt 20 <210> 455 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S17F <400> 455 cacaaaacta cctcaactta 20 <210> 456 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S01R <400> 456 ggtctgttaa tgctctcttg 20 <210> 457 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S02R <400> 457 tcttgttcct tcccaaggtt 20 <210> 458 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S03R <400> 458 tgtgggttca tcaatatcca 20 <210> 459 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S04R <400> 459 gcctccccct tgttgtcaac 20 <210> 460 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S05R &Lt; 400 > 460 gacacataaa gatgttttgg 20 <210> 461 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S06R <400> 461 ccgtgccttg atctgtgcag 20 <210> 462 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S07R <400> 462 ggagcttgca aggttcgctt 20 <210> 463 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S08R <400> 463 tcttttgtct ccatattgcc 20 <210> 464 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S09R <400> 464 gtctggtgct ccagcaaata 20 <210> 465 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S10R <400> 465 tcttagatgc catgattgta 20 <210> 466 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S11R <400> 466 atcctttggt ccagttgtat 20 <210> 467 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S12R <400> 467 caagctctgt gcaagtgttc 20 <210> 468 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S13R <400> 468 gattcagtag tatatgataa 20 <210> 469 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S14R <400> 469 taactgaccc acccctgagt 20 <210> 470 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S15R <400> 470 aagaaagcaa gattagttaa 20 <210> 471 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S16R <400> 471 gacaatcaag gagcaatcaa 20 <210> 472 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HN2S17R <400> 472 gtagtagttt tgctccctaa 20 <210> 473 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> OSM55 <400> 473 tagtagtaga ctcc 14

Claims (13)

A method for identifying whole genome sequences of Hantaan virus (HTNV) in a sample, said method comprising:
(a) preparing a multiplex primer set pool comprising different sets of multiplex primers consisting of a plurality of forward and reverse primer pairs that amplify genomic sequence fragments of mutant 100 to 200 bp long hanthan virus (HTNV) (HTNV) genomic sequence fragments amplified by the one multiplex primer set include 1 to 2 kb of the genomes of the hanthan virus (HTNV), and the 5 'to 3' direction in the fragments The two adjacent fragments comprise an overlapping sequence and the overlapping sequence is common to the 3'-end of the preceding fragment and the 5'-end of the following fragment in lengths of 40 to 60 bp , And when all the fragments amplified by the multiplex primer set pool are overlapped with each other in the 5'-to-3 'direction, the whole genome of the gangrene virus (HTNV) Sequence;
(b) As part of a sample pre-processing process for using next generation sequencing using the prepared primer set pool in a han- tan virus (HTNV) infected sample, a multiplex polymerase chain reaction polymerase chain reaction, mPCR); And
(c) analyzing a germ line virus (HTNV) sequence library comprising the amplified mPCR products with next generation sequencing (NGS)
Lt; / RTI &gt;
Wherein the multiplex primer set pool comprises a forward primer consisting of SEQ ID NO: 1 to SEQ ID NO: 472 and a pair of reverse primers. The method of claim 1,
(B-1), after step (b), a second multiplex primer set pool comprising multiplex primer sets consisting of primer pairs different from the multiplex primer set pool prepared in step (a) ). &Lt; / RTI &gt; The method of claim 1, wherein the sample is obtained from blood, serum, sputum, urine, or living tissue. 2. The method of claim 1, wherein the sample is RNA or cDNA. delete delete The method of claim 1, wherein the method identifies L, M, or S segments of hanthan virus (HTNV). (HTNV) diagnostic kit comprising a multiplex primer set pool, wherein the multiplex primer set pool comprises a forward primer and a reverse primer pair consisting of SEQ ID NOS: 1 to 472, and the primer pair is selected from the group consisting of Hanta virus Lt; RTI ID = 0.0 &gt; L, &lt; / RTI &gt; M and S segments. 9. The kit according to claim 8, wherein the sample is obtained from blood, serum, sputum, urine or living tissue. 9. The kit according to claim 8, wherein the sample is RNA or cDNA. 9. The kit according to claim 8, wherein the kit is performed according to PCR. (HTNV) -induced disease comprising a multiplex primer set pool, wherein the multiplex primer set pool comprises a forward primer and a reverse primer pair consisting of SEQ ID NOS: 1 to 472, 0.0 &gt; L, &lt; / RTI &gt; M and S segments. The composition according to claim 12, wherein the malignant viral (HTNV) -induced disease is hemorrhagic fever with renal syndrome (HFRS) or Hantavirus cardiopulmonary syndrome (HCPS).
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