JP6990836B2 - Detection and / or quantification primer pair, detection and / or quantification probe, and detection and / or quantification kit - Google Patents

Description

The present invention comprises a primer pair for detection and / or quantification of vesicular stomatitis virus Indiana strain, a probe for detection and / or quantification of vesicular stomatitis virus Indiana strain, and detection and / or quantification of vesicular stomatitis virus Indiana strain. Regarding the kit.

Vesicular stomatitis virus (hereinafter abbreviated as "VSV") is a prototype of the Rhabdoviridae family, and there are several serotypes such as Indiana strain. Recombinant VSV can be produced from plasmid DNA and has the advantage of being able to highly express foreign genes inserted into the viral genome. The recombinant VSV Indiana wild strain, which is currently the most frequently used research, has a wide host range and can be easily concentrated by ultracentrifugation.

Recombinant VSV has been studied for many years as a vaccine vector, and one of the most notable recent developments is the development of a vaccine using recombinant VSV against the Ebola virus infection that has been rampant in Africa in recent years. It was also shown to be effective and safe. This vector vaccine is a recombinant virus vaccine prepared by replacing the G gene of VSV with the glycoprotein gene (GP gene) of Ebola virus Sudan strain (SEBOV), Zaire strain (ZEBOV) or Cote d'Ivoire strain (CIEBOV). So, the protective effect in mice has already been reported more than 10 years ago. Due to the outbreak of Ebola virus disease in West Africa, infection control experiments in monkeys and clinical trials in humans are rapidly proceeding, and it is expected to be put into practical use (Non-Patent Documents 1 and 2).

Further, in Patent Document 1, a plasmid vector encoding the VSV G gene is introduced into cells, the VSV G protein is expressed from the VSV G gene, and the cells expressing the VSV G protein are infected with the attenuated VSV. A method for recovering attenuated VSV from a culture is described, in which the cells are allowed to grow and the infected cells are cultured.

Further, Patent Document 2 describes one vaccine or immunoantigenic composition containing one antigen type recombinant vesicular stomatitis virus (rVSV) and one vaccine or immunoantigenic composition containing another antigen type rVSV. The composition and the immune platform having it are described.

By the way, VSV mainly infects livestock such as cattle, horses and pigs, and causes vesicular stomatitis and the like. The pathogenicity to humans is relatively mild and many are asymptomatic, but rarely, influenza-like symptoms such as fever, chills, and myalgia occur. Therefore, a means for monitoring the proliferation and residual levels of recombinant VSV in vivo is required.

Until now, in vivo VSV monitoring has been performed by measuring the infectious titer by the plaque assay method, but it is hard to say that it is a highly accurate method in terms of accuracy, reproducibility and sensitivity. In addition, although some more sensitive nucleic acid test methods (qualitative methods) have been developed in general, it cannot be said that a sufficient method has been established yet, and more accurate quantitative nucleic acid using real-time RT-PCR has been developed. It is necessary to establish an inspection method.

Japanese Patent Publication No. 2011-507515 Japanese Patent Publication No. 2012-528427

Mire, et al., Nature 520: 688-691, 2015 Henao-Restrepo, et al., Lancet 389 (10068): 505-518, 2017

The present invention has been made in view of the above problems, and the detection used in the quantitative nucleic acid test method using real-time RT-PCR for accurately monitoring the recombinant VSV used as a vector in vivo. And / or a primer pair for quantification, a probe for detection and / or quantification, and a kit for detection and / or quantification.

In the primer pair for detecting and / or quantifying the vesicular stomatitis virus Indiana strain according to the present invention, each base sequence of the forward primer and the reverse primer is used.
(A) SEQ ID NOs: 1 and 2,
(B) SEQ ID NOs: 4 and 5,
(C) SEQ ID NOs: 7 and 8,
(D) SEQ ID NOs: 10 and 11 or (e) SEQ ID NOs: 13 and 14.

The base sequence of the probe for detecting and / or quantifying the vesicular stomatitis virus Indiana strain according to the present invention is shown in SEQ ID NO: 3, 6, 9, 12 or 15.

According to the present invention, in vivo monitoring of recombinant VSV can be performed with high accuracy.

It is a figure which shows the outline of the wild type of vesicular stomatitis virus. It is a figure which shows the result of the primary screening of the highly sensitive primer specific to vesicular stomatitis virus. It is explanatory drawing of the secondary screening condition which adopts the TaqMan method in a primer set with a Ct value of 20 or less. It is a figure which shows the result of the secondary screening examined using the wild type strain and the G gene deficient strain. It is a figure which shows the calibration curve using synthetic RNA about the oligoset VSV-77.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating the understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

The present inventors have found a primer pair and a probe capable of detecting and / or quantifying the vesicular stomatitis virus Indiana strain in vivo and the like with high sensitivity and specifically, and thereby used as a vaccine vector and the like in vivo and the like. The vesicular stomatitis virus Indiana strain could be detected and / or quantified.

VSV is a (-) single-stranded RNA type virus belonging to the genus Becyclovirus of the family Rhabdoviridae, and has G protein, which is an enveloped protein, on the membrane surface. VSV infection is established when the G protein binds to phosphatidylserine on the cell membrane surface and then is taken up into the cell by the endocytosis pathway.

The particle morphology of VSV exhibits a characteristic cannonball shape. FIG. 1 is a schematic diagram of the VSV wild type. As shown in this figure, genes encoding five structural proteins (N, P, M, G) in the genome of vesicular stomatitis virus in order from the 3'end are shown. And L gene) are included. The N gene encodes the nucleocapsid protein responsible for the formation of capsids in the genome, while the P (phosphoprotein) and L (large protein) coding sequences constitute subunits of RNA-dependent RNA polymerase. The matrix protein (M) promotes virion maturation and lines the inner surface of the virus particles.

VSV is an attractive candidate as a vector in immunogenic compositions for use in humans due to its many properties, but it also promotes the development of vaccines and therapeutic agents that use VSV as a vector in humans. A means for accurately monitoring the proliferation and residual level of recombinant VSV in vivo is desired.

The present inventor prepared a total of 48 sets of primers for the L gene region of the VSV Indiana strain, performed screening using the full-length DNA of the same strain, and selected 5 sets of primer sets for extremely highly efficient amplification. A probe was prepared for this.

In PCR, DNA polymerase extends a forward primer and a reverse primer to amplify a predetermined region of the double-stranded DNA of the template. The forward primer and the reverse primer consist of a base sequence complementary to the 3'side of each of the double strands of the double-stranded DNA to be amplified.

In PCR, the forward primer is extended from the 5'end to the 3'end by adding deoxynucleotide triphosphate complementary to the template DNA to the 3'end of the forward primer by DNA polymerase. To. In PCR, due to changes in the reaction temperature, the double-stranded template DNA is unwound into a single strand, the forward primer is annealed to the template DNA, an extension reaction is performed by DNA polymerase, and the extended forward primer and template are performed. DNA is unraveled. The same applies to the reverse primer. PCR can amplify the nucleic acid of interest by repeating the above series of reactions.

Specifically, each base sequence of the forward primer and the reverse primer is a primer set shown below.
(a) VSV-77
Forward Primer 5'-CTGCCCTTCATAGGTTTTCG -3' (SEQ ID NO: 1)
Reverse primer 5'-CATCAACCTGGTCAATGCTG -3'(SEQ ID NO: 2)
(b) VSV-126
Forward Primer 5'-CCCGAATTTCAGACTCCTTG -3' (SEQ ID NO: 4)
Reverse primer 5'-GTTTCGGACCAATTCCAGAG -3' (SEQ ID NO: 5)
(c) VSV-31
Forward Primer 5'-ATGAGGCAATTTGCATAGCC -3' (SEQ ID NO: 7)
Reverse primer 5'-CACTGGGCCGTTTGATAACT -3' (SEQ ID NO: 8)
(d) VSV-54
Forward Primer 5'-AAACCCCGAGATAGCCAAGT -3' (SEQ ID NO: 10)
Reverse primer 5'-GCTGGACTCATTCCCATAGC -3' (SEQ ID NO: 11)
(e) VSV-68
Forward Primer 5'-GAGACTCCTTGTGCACCATGT -3' (SEQ ID NO: 13)
Reverse primer 5'-TCCCCGTGAACTAAAGACGT -3' (SEQ ID NO: 14)
Among the above, (a) VSV-77 is particularly preferable. Each of the primer pairs described above can be synthesized by a known method. The primer pair is chemically synthesized by any nucleic acid synthesis method including, for example, a solid phase phosphoramidite method. The primer pair can also be synthesized by the triester method. In addition, various automatic oligonucleotide synthesizers are commercially available, and the primer pair can also be synthesized by the automatic oligonucleotide synthesizer. In addition, a multi-nucleotide synthesis method can also be used as appropriate.

The method of using each of the above-mentioned primer pairs will be described. The primer pair is used in PCR using a VSV-derived nucleic acid as a template. The nucleic acid derived from VSV is, for example, cDNA synthesized by extracting the total RNA of VSV from a serum or plasma sample by a known method and performing a reverse transcription reaction. PCR can be performed with a commercially available PCR device. The conditions for the amplification reaction may be, for example, repeating 45 cycles of 50 ° C. for 20 minutes, 95 ° C. for 15 minutes, then 94 ° C. for 45 seconds, and 60 ° C. for 45 seconds.

By quantifying the amplification product by PCR, VSV contained in the sample can be detected and / or quantified. The method for quantifying the amplified product is not particularly limited, such as an intercalation method and a hybridization method. In the intercalation method, for example, SYBR Green may be used as a dye that is specifically inserted into double-stranded DNA and emits fluorescence. In the hybridization method, a probe in which a fluorescent dye is bound to an oligonucleotide having a base sequence complementary to the base sequence of the amplification product may be used. TaqMan® is particularly preferred as the probe. In order to quantify the amplified product, a real-time quantitative PCR method in which the amplified product by PCR is measured over time is preferably used. Further, the amplified product may be quantified by agarose gel electrophoresis or the like. The hybridization condition in the hybridization method is a stringent condition in which the probe hybridizes with the amplification product but does not hybridize with the nucleic acid consisting of other base sequences.

The probe according to this embodiment is suitable for quantifying the amplification product amplified by the primer pair. The base sequence of each probe is shown below. The probe can be synthesized by a known method as well as the primer pair.
(a) VSV-77
Probe 5'-CCATGGTGGGTTCG -3' (SEQ ID NO: 3)
(b) VSV-126
Probe 5'-CCCAATCGGGAACTG -3' (SEQ ID NO: 6)
(c) VSV-31
Probe 5'-CGAAAATGGAATAACC -3' (SEQ ID NO: 9)
(d) VSV-54
Probe 5'-CTCACATAGACAAGCTAG -3' (SEQ ID NO: 12)
(e) VSV-68
Probe 5'-CAGGGTTCAATTATG -3' (SEQ ID NO: 15)
Among the above, (a) VSV-77 is particularly preferable. The probe is preferably labeled with a fluorescent dye, radioactive material, or the like. Fluorescein and fluorescein derivatives such as FAM, VIC, JOE, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid, coumarin and coumarin derivatives, lucifer yellow, Texas red, tetramethylrhodamine, 6 as fluorescent dyes. Examples thereof include -carboxyfluorescein, tetrachloro-6-carboxyfluorescein, 5-carboxyrhodamine, and cyanine dyes.

As a probe labeled with a fluorescent dye, a probe in which a fluorescent dye is bound to the 5'end and a quencher that induces quenching of the fluorescent dye by the principle of fluorescence resonance energy transfer is bound to the 3'end is particularly preferable. Quenchers include tetramethylrhodamine [TAMRA], 4'-(4-dimethylaminophenylazo) benzoic acid, 4-dimethylaminophenylazophenyl-4'-maleimide, tetramethylrhodamine, carboxytetramethylrhodamine and BHQ dyes. And so on.

The base sequence of the primer pair and the probe according to the present embodiment may be a base sequence in which one to several bases are deleted, substituted or added in the base sequence specified above. Further, the base sequence of the primer pair and the probe may be a base sequence having 90% or more sequence identity with the base sequence specified above. Further, the base sequence of the primer pair and the probe may be a base sequence capable of hybridizing under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence specified above.

Next, the kit for detecting and / or quantifying the vesicular stomatitis virus Indiana strain according to the present embodiment includes the above-mentioned primer pair and probe.

Specifically, the first detection and / or quantification kit comprises the following forward and reverse primers and the following probe, as shown in Table 1 below.
(a) VSV-77
Forward Primer 5'-CTGCCCTTCATAGGTTTTCG -3' (SEQ ID NO: 1)
Reverse primer 5'-CATCAACCTGGTCAATGCTG -3'(SEQ ID NO: 2)
Probe 5'-CCATGGTGGGTTCG -3' (SEQ ID NO: 3)
The second detection and / or quantification kit comprises the following forward and reverse primers and the following probe, as shown in Table 1 below.
(b) VSV-126
Forward Primer 5'-CCCGAATTTCAGACTCCTTG -3' (SEQ ID NO: 4)
Reverse primer 5'-GTTTCGGACCAATTCCAGAG -3' (SEQ ID NO: 5)
Probe 5'-CCCAATCGGGAACTG -3' (SEQ ID NO: 6)
The third detection and / or quantification kit comprises the following forward and reverse primers and the following probe, as shown in Table 1 below.
(c) VSV-31
Forward Primer 5'-ATGAGGCAATTTGCATAGCC -3' (SEQ ID NO: 7)
Reverse primer 5'-CACTGGGCCGTTTGATAACT -3' (SEQ ID NO: 8)
Probe 5'-CGAAAATGGAATAACC -3' (SEQ ID NO: 9)
The fourth detection and / or quantification kit comprises the following forward and reverse primers and the following probe, as shown in Table 1 below.
(d) VSV-54
Forward Primer 5'-AAACCCCGAGATAGCCAAGT -3' (SEQ ID NO: 10)
Reverse primer 5'-GCTGGACTCATTCCCATAGC -3' (SEQ ID NO: 11)
Probe 5'-CTCACATAGACAAGCTAG -3' (SEQ ID NO: 12)
The fifth detection and / or quantification kit comprises the following forward and reverse primers and the following probe, as shown in Table 1 below.
(e) VSV-68
Forward Primer 5'-GAGACTCCTTGTGCACCATGT -3' (SEQ ID NO: 13)
Reverse primer 5'-TCCCCGTGAACTAAAGACGT -3' (SEQ ID NO: 14)
Probe 5'-CAGGGTTCAATTATG -3' (SEQ ID NO: 15)

According to the present invention, a recombinant virus in which the G gene of the vesicular stomatitis virus Indiana strain has been replaced (or has not been replaced) that proliferates in the living body or remains in the living body is detected and quantified with high sensitivity. It is possible. Specifically, the vesicular stomatitis virus Indiana strain as a vaccine vector of a recombinant virus vaccine in which the G gene of the vesicular stomatitis virus Indiana strain is replaced with the glycoprotein gene of Ebola virus can be detected and quantified with high sensitivity.

A total of 48 sets of primers were prepared for the L gene region of the Indiana wild strain, and screening was performed by the SYBR Green method using the full-length DNA of the strain. SYBR Green is a type of intercalator that binds to DNA, binds to double-stranded DNA synthesized by PCR, and fluoresces when irradiated with excitation light. By measuring this fluorescence intensity, the production amount of the PCR amplification product can be monitored. For PCR, Applied Biosystems 7500 fast (manufactured by Applied Biosystems) was used. The attached analysis software (7500 software v2.3) was used to analyze the data. The Power SYBR Green RNA-to-CT 1-Step Kit (manufactured by Thermo Fisher) was used for the primary screening by the SYBR Green method. The PCR conditions were the recommended conditions for the kit. The results of the primary screening are shown in FIG. "Ct" indicates the number of cycles when the fluorescence intensity exceeds the detection threshold.

In the secondary screening, the hybridization method (TaqMan® method) was adopted for the primer set showing excellent amplification efficiency with a Ct value of 20 or less in the primer set shown in FIG. For screening, TaqMan (registered trademark) RNA-to-CT 1-Step Kit (manufactured by Thermo Fisher) was used. The PCR conditions were the recommended conditions for the kit. The primer concentration was the final 400 nM for both forward and reverse, and the probe concentration was the final 200 nM. Tm was around 60 ° C for all five sets. The results are shown in FIG. The fluorescent dye at the 5'end was FAM, and the quencher at the 3'end was BHQ.

The above oligoset was examined by the TaqMan method using wild-type strains and G gene-deficient strains. Here, VSVΔG RNA is obtained by infecting BHK-G cells with VSVΔG (MOI: 1.0), collecting the culture supernatant after 24 hours, and using the QIA amp Viral RNA Mini Kit (Qiagen) from the collected virus solution. RNA was purified according to the protocol, and the purified RNA was used as a template for PCR. The results are shown in Table 2 and FIG. 4 below. VSV-77 was decided as the combination with the best sensitivity and specificity. As shown in FIG. 4, not only the plasmid DNA of pVSV-XN2 (recombinant VSV wild strain) but also RNA purified from the virus particles (recombinant VSVΔG) itself could be amplified. This is because VSVΔG carries the L gene in its genome, similar to the wild-type strain. In addition, there was no non-specific amplification in the DNA / RNA of other viruses, and only the DNA / RNA of recombinant VSV (wild strain and ΔG virus) could be specifically amplified.

A calibration curve was prepared for this oligoset VSV-77 using synthetic RNA. The results are shown in FIG. The linearity of the calibration curve was evaluated by the correlation coefficient R ² , where R ² was 0.999. The slope of the calibration curve was also within the appropriate range.

It can be used to detect and quantify vesicular stomatitis virus.

SEQ ID NO: 1,2,4,5,7,8,10,11,13,14: Primer SEQ ID NO: 3,6,9,12,15: Probe

Claims (4)

Each base sequence of the forward primer and the reverse primer
(A) shown in SEQ ID NOs: 1 and 2 ,
Primer pair for detection and / or quantification of vesicular stomatitis virus Indiana strain. The primer pair for detection and / or quantification according to claim 1, wherein the vesicular stomatitis virus Indiana strain is a recombinant virus in which the G gene of the vesicular stomatitis virus Indiana strain is substituted (or not substituted). The vesicular stomatitis virus Indiana strain is the vesicular stomatitis virus Indiana strain as a vaccine vector of a recombinant virus vaccine in which the G gene of the vesicular stomatitis virus Indiana strain is replaced with the glycoprotein gene of Ebola virus according to claim 2. A pair of detection and / or quantification primers. A first primer pair consisting of a forward primer whose base sequence is shown in SEQ ID NO: 1 and a reverse primer whose base sequence is shown in SEQ ID NO: 2, and a first probe whose base sequence is shown in SEQ ID NO: 3. 1 detection and / or quantification kit ,
A kit for detecting and / or quantifying the vesicular stomatitis virus Indiana strain.

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