Patent Issued for Pretreatment Method And Nucleic-Acid-Extracting Kit Usable For The Same (USPTO 10,316,350)

2019 JUN 25 (NewsRx) -- By a News Reporter-Staff News Editor at Hospital & Nursing Home Daily -- According to news reporting originating from Alexandria, Virginia, by NewsRx journalists, a patent by the inventors Yamakawa, Kazutomi (Saga, Japan); Nagano, Takashi (Saga, Japan), filed on September 2, 2015, was published online on June 24, 2019.

The assignee for this patent, patent number 10,316,350, is Mizuho Medy Co. Ltd. (Saga, Japan).

Reporters obtained the following quote from the background information supplied by the inventors: “The present invention relates to a pretreatment method of, prior to a nucleic-acid-amplifying process, extracting nucleic-acid by means of a solid-phase extraction method.

“Herein, the nucleic-acid-amplifying process is performed in order to detect and/or identify the base sequence of target nucleic-acid.

“More particularly, the present invention is capable of realizing processes according to a POCT mode without any special physical and chemical instruments.

“The word of ‘POCT’ stands for ‘Point of Care Testing’, and means an inspection performed near a subject and/or by the subject himself/herself.

“The Japan society for clinical laboratory automation has defined, in POCT guidelines thereof, that the POCT is ‘an inspection for contributing to improvement in items including: prompt and appropriate medical treatment and nursing; prevention of disease; quality of medical cares, such as health care administration; QOL (Quality of life); and satisfaction of the cares.’

“In recent years, the life science field has remarkably developed, and nucleic-acid (such as DNA and RNA)-analyzing techniques have been widely used.

“The nucleic-acid-analyzing techniques have been used widely, for example, in: biological fields (species identification, studies of the origins thereof, or the like.); medical fields (diagnosis of diseases, or the like.); and other fields close to daily life (confirmation of the food safety, or the like.).

“Target nucleic-acid may include, for example, specific gene sequences of foreign genes that do not exist in an owner of the sample and other specific base sequences. ‘Genetic screening’ is a technique that amplifies and analyzes the target nucleic-acid by means of a PCR method and/or an isothermal nucleic-acid-amplifying method among nucleic-acid-analyzing techniques.

“Whereas, culture tests and immunological examinations of inspections by means of antigens and/or antibodies belong to conventional infectious disease diagnostic techniques.

“In disease diagnosis in the medical fields, particularly in early infectious disease diagnosis, the genetic screening has higher sensitivity than the conventional infectious disease diagnostic techniques, or the like.

“Utilizing the genetic screening enables to detect and/or identify almost all pathogenic microorganisms including: bacteria; fungi; protozoa; and viruses, which may be responsive for causing infectious diseases in human beings.

“Accordingly, the genetic screening is considered to be a useful inspection method for early diagnosis of the infectious diseases, and has been frequently used in facilities with exclusive inspecting rooms and/or laboratories, such huge hospitals (e.g. base hospitals), health laboratories, research establishments (e.g. universities and companies), other institutions with many staffs, or the like.

“In ‘2012-Clinical-examination-market No. 3/bacteria/gene/POC/biopsy’, the Fuji Keizai Co., LTD. has pointed out ‘It is expected that the market of the genetic screening will expand at an annual ratio of 2 through 3% also after 2013.’ It is expected that the market of the genetic screening will increase the importance thereof with respect to not only the early diagnosis of the infectious diseases but also economic activities.

“The genetic screening is, however, hardly used in small medical facilities often visited by ordinary people (e.g. private hospitals and/or clinics in the town).

“The reason why is as follows.

“Firstly, the genetic screening requires complicated and time-consuming operation before performing the same, such as reagent preparation, nucleic-acid extraction, or the like, which forces a heavy burden on the small medical facilities.

“Secondly, performing the genetic screening also requires physical and chemical equipment and automation equipment. The physical and chemical equipment includes a centrifugal machine and a micro pipette for extracting nucleic-acid. Thereby, both initial investment costs and maintenance costs must be higher.

“Thirdly, it cannot be said that doctors, nurses, or the like in the small medical facilities are fully conversant with the genetic screening.

“Meanwhile, testing kits not related to the genetic screening but corresponding to the POCT (for example, kits according to an labeled antibody method, an immunoturbidimetric method, a latex agglutination method, a immunochromatography method, or the like.) are widely used also in the small medical facilities.

“According to the summary announced by the Japan Ministry of Health, Labor and Welfare in December 2009, actual shipped production of rapid examination kits against influenza antigen in the previous season have reached about 13 million tests.

“The genetic screening using target nucleic-acid has more sensitivity than an immunological examination, and is very effective in rapid diagnosis of early infectious disease.

“Unfortunately, neither a POCT kit applicable for the genetic screening nor another kit pursuant the same exists at present.

“For this reason, in the small medical facilities, the genetic screening cannot be performed. Alternatively, specimens are collected to be left for inspection by one of external facilities. Therefore, genetic screening results cannot be obtained quickly.

“If POCT kits applicable for the genetic screening and a method for the same have been developed, also in the small medical facilities, the genetic screening can be performed, thereby obtaining extremely high utility.

“Herein, the present genetic screening method can be roughly divided into the following three processes.

“A first process (pretreatment) performs: after having collected a specimen, exposing nucleic-acid conjugated by shells and/or membrane of protein in the specimen therefrom; and washing and separating contaminants, such as protein, by means of organic solvent, a solid-phase carrier, or the like to isolate the nucleic-acid only.

“A second process (amplification of the target nucleic-acid) performs: utilizing a template of the separated nucleic-acid; and amplifying the target nucleic-acid according to a nucleic-acid amplification reaction method, such as PCR, LAMP reaction, or the like.

“A third process (detection and analysis) performs: during and/or after the amplification reaction, using the amplified target nucleic-acid and/or markers conjugating to the same to carry out a qualitative step and/or a quantitative step related thereto.

“There is automating equipment for easily performing the first process.

“However, the automating equipment for the first process is too expensive, and has been hardly spread at present.

“Practically in many cases, the pretreatment tends to be performed by means of a manual method capable of carried out more cheaply than the automating equipment.

“However, since the manual method requires a mastery of arts, such as how to handle equipment (e.g. a centrifugal machine and/or a micropipette), the sample, or the like, the operational burden on the operator must be heavy.

“Neither a method nor a kit is known which can simplify the manual method so as to be used in the POCT mode.

“As for one representative solid-phase extraction method used in the automating equipment, there is a method reported by Boom et al (hereinafter, called as the ‘BOOM method’. See, Reference 1 and Reference 5.).

“The BOOM method is the solid-phase extraction method for isolating nucleic-acid from a biological sample according to a principle based on chaotropic effects (See, Reference 6.) caused by a phenomenon that the nucleic-acid absorbs silica beads in the presence of a chaotropic agent.

“According to the BOOM method, without making nucleic-acid elute from a solid-phase carrier, the nucleic-acid can be added to PCR solution together with the solid-phase carrier (See, Reference 2.), and a part of elution process can also be simplified. The BOOM method, however, assumes that environment for performing the extraction of nucleic-acid, such as an inspecting room, has been prepared.

“Reference 3 recites as follows. Namely, ‘An object according to the present invention is to provide: a method of, without using organic solvent, simply, in a short time, further safely, and with high reproducibility extracting ribonucleic acid from biological material; and a reagent for the method. Dissimilar to deoxyribonucleic acid, upon washing with low salt concentration buffer solution containing no organic solvent (e.g. ethanol, or the like.) after having made ribonucleic acid absorb a solid-phase carrier, the ribonucleic acid hardly elutes from the solid-phase carrier. Once well-heated, however, the ribonucleic acid is promoted to elute there-from. A second process in the present invention is a process of washing a solid-phase carrier on which ribonucleic acid have absorbed due to a first absorption process by means of wash fluid composed of low salt concentration buffer solution in order to remove chaotropic material, or the like there-from. Herein, the means buffer solution containing neither organic solvent (e.g. ethanol, or the like.) nor chaotropic material. It is preferable that the buffer solution is Tris system buffer solution. However, the present invention is never limited to the Tris system buffer solution. The means a level of salt concentration that does not affect enzyme reaction (e.g. RT-PCR, or the like.) even when this buffer solution remains in the third elution process, and also includes water itself solely. Preferably in the present invention, 100 or less [mM] of buffer solution may be used. This solution may contain a surface active agent and pH thereof is not limited in particular. In the present invention, it is necessary to promote the elution by heating. Heating temperature is not limited in particular, as long as it does not cause adverse effects on the ribonucleic acid, but may be preferably 50 through 70 [Centigrade]. Hearing time is about 30 [s] through 10 [min]. The ribonucleic acid eluted in this way can be directly used for the enzyme reaction by means of reverse transcriptase or the like without performing: dialysis; desalting (e.g. according to an ethanol precipitation method, or the like.); or concentration operation (partially omitted)’.

“Reference 4 recites, ‘However, the present inventors have found that, in the elution process (3), making nucleic-acid elute with water and/or low salt concentration solution at 80 or more [Centigrade] enables to extract DNA, thereby having completed the present invention.’

“The methods recited in these References have the following disadvantages, and are difficult to be turned into the POCT mode.

“Firstly, they require complicated operation with high difficulty, because a few through some hundreds [.mu.l] of solution must be collected and added, contamination must be prevented according to accurate operation of pipets, and hazardous reagent must be dealt with.

“Secondly, they also require: washing the target several times with wash fluid containing salt and/or organic solvent; and surely removing the organic solvent according to drying process therefrom.

“They further require: performing the drying process while taking care of fixation between the nucleic-acid and the adsorbing carrier caused by excessive drying. Consequently, long time and great effort must be paid.

“Thirdly, initial costs for preparing operation environment must be high, because they require: operation using the centrifugal machine and the micropipette; and an installation site and/or a working space for used equipment. Reference 1: Japanese registered patent No. 2680462 Reference 2: Japanese patent application Laid-open No. 10-72485 Reference 3: Japanese registered patent No. 3812696 Reference 4: Japanese patent application Laid-open No. 2014-30364 Reference 5: R Boom et al, J. Clin Microbiol, 28 (3), 495-503 (1990) Reference 6: B Vogelstein and D Gillespie, PNAS, 76 (2), 615-619 (1979)”

In addition to obtaining background information on this patent, NewsRx editors also obtained the inventors’ summary information for this patent: “In view of the above background, an object according to the present invention is to provide a method capable of realizing pretreatment process of genetic screening according to a POCT mode.

“A first aspect of the present invention provides: a pretreatment method, comprising: making a sample, extraction liquid for extracting nucleic-acid contained in the sample, silica particles, and a filtering material contact with each other; making the filtering material carry composite material of the nucleic-acid and the silica particles thereon; and then delivering the filtering material to a nucleic-acid-amplifying process by means of reaction solution for amplifying nucleic-acid, wherein particle diameters of the silica particles and concentration of the silica particles in the reaction solution for amplifying nucleic-acid are set up within a predetermined range, thereby allowing, in advance of the nucleic-acid-amplifying process, to omit both a drying process and an elution process.

“A second aspect of the present invention provides: in addition to the first aspect, the pretreatment method wherein the predetermined range is defined as follows: the concentration of the silica particles is 0.0625 through 4 [.mu.g/.mu.l]; an average particle diameter of the silica particles is 0.01 through 100 [.mu.m]; and a surface area calculated based on the average particle diameter is 1.times.10.sup.4 through 1.times.10.sup.8 [.mu.m].

“A third aspect of the present invention provides: in addition to the first aspect, the pretreatment method wherein the predetermined range is defined as follows: the concentration of the silica particles is 0.0625 through 1 [.mu.g/.mu.l]; an average particle diameter of the silica particles is 0.01 through 10 [.mu.m]; and a surface area calculated based on the average particle diameter is 1.times.10.sup.5 through 5.times.10.sup.7 [.mu.m.sup.2].

“A fourth aspect of the present invention provides: in addition to the first aspect, the pretreatment method further comprising: a first extraction process of adding the sample to the extraction liquid to extract the nucleic-acid contained in the sample; a first absorption process of: making the silica particles contact with the extracted nucleic-acid to obtain the composite material of the nucleic-acid and the silica particles; and making the composite material contact with the filtering material; and a first washing process of: washing the composite material and the filtering material with purified water; and delivering the washed composite material and the washed filtering material to the nucleic-acid-amplifying process.

“A fifth aspect of the present invention provides: in addition to the first aspect, the pretreatment method wherein, prior to the delivering the filtering material to the nucleic-acid-amplifying process, the composite material is separated from the filtering material, and then the separated composite material is delivered to the nucleic-acid-amplifying process.

“A sixth aspect of the present invention provides: in addition to the fourth aspect, the pretreatment method wherein the first absorption process and the first washing process are performed at the same time.

“A seventh aspect of the present invention provides: in addition to the fourth aspect, the pretreatment method wherein the first extraction process and the first absorption process are performed at the same time.

“An eighth aspect of the present invention provides: in addition to the fourth aspect, the pretreatment method wherein the first extraction process, the first absorption process, and the first washing process are performed at the same time.

“A ninth aspect of the present invention provides: in addition to the first aspect, the pretreatment method further comprising: a second extraction process of adding the sample to the extraction liquid to extract the nucleic-acid contained in the sample; a second absorption process of: making the extracted nucleic-acid contact with the filtering material carrying the silica particles thereon to obtain the composite material of the nucleic-acid and the silica particles; and making the filtering material carry the obtained composite material thereon; and a second washing process of: washing the carried composite material and the filtering material with purified water; and delivering the washed composite material and the washed filtering material to the nucleic-acid-amplifying process.

“A tenth aspect of the present invention provides: in addition to the ninth aspect, the pretreatment method wherein, prior to the delivering the filtering material to the nucleic-acid-amplifying process, the composite material are separated from the filtering material, and then the separated composite material is delivered to the nucleic-acid-amplifying process.

“An eleventh aspect of the present invention provides: in addition to the ninth aspect, the pretreatment method wherein the second absorption process and the second washing process are performed at the same time.

“Effect of the Invention

“With the methods according to the present invention, the following effects can be obtained.

“Firstly, the extraction process of nucleic-acid in genetic screening can be simple, easy, safe, rapid, and less expensive, and capable of realizing the process of according to the POCT mode.

“Secondly, the combination of safe, simple, and easy operation enables to remarkably shorten operation time comparing with the conventional methods, and further the extraction of nucleic-acid can be performed at a low cost.

“This is because the purified water is used as the wash fluid. Accordingly, it is unnecessary to prepare a plurality of kinds of organic solvent. It is also unnecessary to use the plurality of kinds of organic solvent while distinguishing thereof from each other. Furthermore, the organic solvent itself is unnecessary, and the operation is easy and safe.

“Thirdly, since the organic solvent itself is unnecessary, both drying operation upon the solid-phase carrier and the elution process of nucleic-acid can be omitted.

“In short, immediately after the washing process, the target can be delivered to the nucleic-acid amplification reaction.

“In addition, since the time of transfer of the solution can be remarkably reduced, environmental risks, such as splashes of nucleic-acid and/or cross contamination can be also reduced.”

The claims supplied by the inventors are:

“What is claimed is:

“1. A pretreatment method of extracting nucleic-acid by means of a solid-phase extraction method prior to a nucleic-acid-amplifying process, the pretreatment method comprising: making a sample, extraction liquid for extracting nucleic-acid contained in the sample, silica particles, and a filtering material contact with each other; making the filtering material carry composite material of the nucleic-acid and the silica particles thereon; and then delivering the filtering material to a nucleic-acid-amplifying process, the nucleic-acid-amplifying process using a reaction solution for amplifying nucleic-acid, wherein particle diameters of the silica particles and concentration of the silica particles in the reaction solution for amplifying nucleic-acid are set up within a predetermined range; wherein the predetermined range allows, in advance of the nucleic-acid-amplifying process, omission of both a drying process and an elution process; wherein the predetermined range is defined as follows: 5% through 38% of the nucleic-acid is recovered from the sample when based on 100% of nucleic-acid contained in the sample; the concentration of the silica particles is 0.0625 .mu.g/.mu.l through 4 .mu.g/.mu.l; an average particle diameter of the silica particles is 0.01 .mu.m through 100 .mu.m; and a surface area calculated based on the average particle diameter is 1.times.10.sup.4 .mu.m.sup.2 through 1.times.10.sup.8 .mu.m.sup.2; and the pretreatment method further comprises: a first extraction process of adding the sample to the extraction liquid to extract the nucleic-acid contained in the sample; a first absorption process of: making the silica particles contact with the extracted nucleic-acid to obtain the composite material of the nucleic-acid and the silica particles; and making the composite material contact with the filtering material; a first washing process of: washing the composite material and the filtering material with purified water; and delivering the washed composite material and the washed filtering material under a wet condition to the nucleic-acid-amplifying process; and wherein, prior to the delivering the filtering material to the nucleic-acid-amplifying process, the composite material is collected with the filtering material in a manner such that the composite material is on the filtering material.

“2. The pretreatment method as defined in claim 1, wherein, prior to the delivering the filtering material to the nucleic-acid-amplifying process, the composite material is separated from the filtering material, and then the separated composite material is delivered to the nucleic-acid-amplifying process.

“3. The pretreatment method as defined in claim 1, wherein the first absorption process and the first washing process are performed at the same time.

“4. The pretreatment method as defined in claim 1, wherein the first extraction process and the first absorption process are performed at the same time.

“5. The pretreatment method as defined in claim 1, wherein the first extraction process, the first absorption process, and the first washing process are performed at the same time.

“6. A pretreatment method of extracting nucleic-acid by means of a solid-phase extraction method prior to a nucleic-acid-amplifying process, the pretreatment method comprising: making a sample, extraction liquid for extracting nucleic-acid contained in the sample, silica particles, and a filtering material contact with each other; making the filtering material carry composite material of the nucleic-acid and the silica particles thereon; and then delivering the filtering material to a nucleic-acid-amplifying process, the nucleic-acid-amplifying process using a reaction solution for amplifying nucleic-acid, wherein particle diameters of the silica particles and concentration of the silica particles in the reaction solution for amplifying nucleic-acid are set up within a predetermined range; wherein the predetermined range allows, in advance of the nucleic-acid-amplifying process, omission of both a drying process and an elution process; wherein the predetermined range is defined as follows: 5% through 38% of the nucleic-acid is recovered from the sample when based on 100% of nucleic-acid contained in the sample: the concentration of the silica particles is 0.0625 .mu.g/.mu.l through 1 .mu.g/.mu.l; an average particle diameter of the silica particles is 0.01 .mu.m through 10 .mu.m; and a surface area calculated based on the average particle diameter is 1.times.10.sup.5 .mu.m2 through 5.times.10.sup.7 .mu.m.sup.2; and the pretreatment method further comprises: a first extraction process of adding the sample to the extraction liquid to extract the nucleic-acid contained in the sample; a first absorption process of: making the silica particles contact with the extracted nucleic-acid to obtain the composite material of the nucleic-acid and the silica particles; and making the composite material contact with the filtering material; a first washing process of: washing the composite material and the filtering material with purified water; and delivering the washed composite material and the washed filtering material under a wet condition to the nucleic-acid-amplifying process; and wherein, prior to the delivering the filtering material to the nucleic-acid-amplifying process, the composite material is collected with the filtering material in a manner such that the composite material is on the filtering material.

“7. The pretreatment method as defined in claim 6, wherein, prior to the delivering the filtering material to the nucleic-acid-amplifying process, the composite material is separated from the filtering material, and then the separated composite material is delivered to the nucleic-acid-amplifying process.

“8. The pretreatment method as defined in claim 6, wherein the first absorption process and the first washing process are performed at the same time.

“9. The pretreatment method as defined in claim 6, wherein the first extraction process and the first absorption process are performed at the same time.

“10. The pretreatment method as defined in claim 6, wherein the first extraction process, the first absorption process, and the first washing process are performed at the same time.”

For more information, see this patent: Yamakawa, Kazutomi; Nagano, Takashi. Pretreatment Method And Nucleic-Acid-Extracting Kit Usable For The Same. U.S. Patent Number 10,316,350, filed September 2, 2015, and published online on June 24, 2019. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=10,316,350.PN.&OS=PN/10,316,350RS=PN/10,316,350

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