Enzyme Preparation Containing Thermostable DNA Polymerase, Method for Producing Same, and Method for Detecting Subject Organism to be Detected

Abstract

Disclosed is a thermostable DNA polymerase preparation which can illimitably reduce the risk of false positivity in the detection of a subject microorganism utilizing a gene amplification reaction and therefore enables the selective amplification of DNA for detecting the subject microorganism even when the amount of the subject microorganism is small and therefore the amount of DNA collected therefrom is extremely small, and can be produced at a reduced cost. Also disclosed is a method for quantifying or quantifying/identifying a subject organism to be detected rapidly, conveniently and with high sensitivity using the preparation of the present invention.

1 . A thermostable DNA polymerase preparation, wherein:
 (1) the thermostable DNA polymerase has contamination with 10 fg or less of bacterially-derived nucleic acid other than a gene encoding the thermostable DNA polymerase based on 1 unit of the thermostable DNA polymerase, and   (2) no amplification products of the bacterially-derived nucleic acid are detected even when 32 cycles or more of gene amplification reaction are performed in the preparation under conditions containing no template using primers capable of amplifying only the bacterially-derived nucleic acid other than the gene encoding the thermostable DNA polymerase.   
     
     
         2 . A production method for a thermostable DNA polymerase preparation, comprising the steps of:
 (1) culturing transformant eukaryotic cells carrying a gene encoding the thermostable DNA polymerase,   (2) obtaining an extract containing the thermostable DNA polymerase from the cultured transformant eukaryotic cells, followed by subjecting the extract to heat treatment, or   subjecting the cultured transformant eukaryotic cells to heat treatment, followed by obtaining an extract containing the thermostable DNA polymerase from the heat-treated transformant cells.   
     
     
         3 . The production method according to  claim 2 , wherein the heat treatment is performed at a temperature of 50° C. or more. 
     
     
         4 . The production method according to  claim 2 , wherein the gene encoding the thermostable DNA polymerase used for the transformation is derived from a thermophilic bacterium or a hyperthermophilic bacterium. 
     
     
         5 . The production method according to  claim 2 , wherein the gene encoding the thermostable DNA polymerase comprises DNA consisting of the base sequence of SEQ NO:1. 
     
     
         6 . The production method according to  claim 2 , wherein the eukaryotic cells are selected from the group consisting of fungi, plant cells, insect cells, and animal cells. 
     
     
         7 . The production method according to  claim 2 , wherein the preparation is that in which:
 (1) the thermostable DNA polymerase has contamination with 10 fg or less of bacterially-derived nucleic acid other than the gene encoding the thermostable DNA polymerase based on 1 unit of the thermostable DNA polymerase, and   (2) no amplification products of the bacterially-derived nucleic acid are detected even when 32 cycles or more of gene amplification reaction are performed in the preparation under conditions containing no template using primers capable of amplifying only the bacterially-derived nucleic acid other than the gene encoding the thermostable DNA polymerase.   
     
     
         8 . A thermostable DNA polymerase preparation comprising the extract obtained by the production method according  claim 2  or a purified preparation thereof. 
     
     
         9 . A set comprising a thermostable DNA polymerase preparation according to  claim 1 . 
     
     
         10 . The set according to  claim 9 , wherein the set comprises primers for amplification of a nucleic acid. 
     
     
         11 . The set according to  claim 10 , wherein the nucleic acid is bacterial DNA. 
     
     
         12 . The set according to  claim 11 , wherein the bacterial DNA is from an  eubacterium  or an ancient bacteria. 
     
     
         13 . A set for quantifying and/or identifying a subject organism to be detected contained in a sample, comprising a thermostable DNA polymerase preparation for amplifying nucleic acid prepared from the sample and primers for amplifying an intended gene specific for the subject organism to be detected,
 wherein the thermostable DNA polymerase preparation is any one of the following (a) and (b):   (a) a thermostable DNA polymerase preparation produced using eukaryotic cells as a host; and   (b) a thermostable DNA polymerase preparation, wherein:   (b-1) the thermostable DNA polymerase has contamination with 10 fg or less of bacterially-derived nucleic acid other than a gene encoding the thermostable DNA polymerase based on 1 unit of the thermostable DNA polymerase, and   (b-2) no amplification products of the bacterially-derived nucleic acid are detected even when 32 cycles or more of gene amplification reaction are performed in the preparation under conditions containing no template using primers capable of amplifying only the bacterially-derived nucleic acid other than a gene encoding the thermostable DNA polymerase.   
     
     
         14 . The set according to  claim 13  for quantifying and/or identifying a subject organism to be detected contained in a sample, comprising:
 one of the following (a) and (b): 
 (a) a thermostable DNA polymerase preparation produced using eukaryotic cells as a host; and 
 (b) a thermostable DNA polymerase preparation, wherein: 
 (b-1) the thermostable DNA polymerase has contamination with 10 fg or less of bacterially-derived nucleic acid other than a gene encoding the thermostable DNA polymerase based on 1 unit of the thermostable DNA polymerase, and 
 (b-2) no amplification products of the bacterially-derived nucleic acid are detected even when 32 cycles or more of gene amplification reaction are performed in the preparation under conditions containing no template using primers capable of amplifying only the bacterially-derived nucleic acid other than a gene encoding the thermostable DNA polymerase, 
 for amplifying nucleic acid prepared from the sample, 
 a thermostable DNA polymerase preparation for amplifying nucleic acid prepared from the sample, produced using bacterial cells as a host, and 
 primers for amplifying an intended gene specific for the subject organism to be detected. 
 
     
     
         15 . The set according to  claim 14 , wherein the primers are the following primers (B), (F) and (M):
 (B) a primer set capable of amplifying a plurality of regions of the 16S rRNA gene of all bacteria and primers containing all or ⅓ or more of each of the base sequences of the primers,   (F) a primer set capable of amplifying a plurality of regions of the 18S rRNA gene of all fungi and primers containing all or ⅓ or more of each of the base sequences of the primers, and   (M) a primer set specifically amplifying an antibiotic resistance gene reflecting an epidemic of the time such as a mec A gene exhibiting methicillin resistance.   
     
     
         16 . The set according to  claim 9 , wherein said thermostable DNA polymerase preparation is the only thermostable DNA polymerase preparation. 
     
     
         17 . The set according to  claim 16 , further comprising at least one of a buffer solution or water. 
     
     
         18 . The set according to  claim 16 , wherein the set comprises first primers for amplification of a first nucleic acid. 
     
     
         19 . The set according to  claim 18 , wherein the first nucleic acid is bacterial DNA. 
     
     
         20 . The set accordingly to  claim 19 , wherein the bacterial DNA is from an  eubacterium  or an ancient bacteria. 
     
     
         21 . The set according to  claim 19 , wherein the set comprises second primers for amplification of a second nucleic acid. 
     
     
         22 . The set according to  claim 20 , wherein the second nucleic acid is fungal DNA. 
     
     
         23 . The set according to  claim 9 , wherein said set comprises the thermostable DNA polymerase preparation according to  claim 1  as a first thermostable DNA polymerase preparation and further comprises a second thermostable DNA polymerase preparation. 
     
     
         24 . The set according to  claim 23 , further comprising at least one of a buffer solution or water. 
     
     
         25 . The set according to  claim 9 , wherein the set comprises first primers for amplification of a first nucleic acid and second primers for amplification of a second nucleic acid. 
     
     
         26 . The set according to  claim 25 , wherein said first nucleic acid is bacterial DNA and said second nucleic acid is fungal DNA. 
     
     
         27 . The set according to  claim 26 , wherein said bacterial DNA is from an  eubacterium  or an ancient bacterium. 
     
     
         28 . The set according to  claim 9 , further comprising at least one of a buffer solution or water. 
     
     
         29 . The set according to  claim 16 , wherein the only thermostable DNA polymerase preparation is produced using eukaryotic cells as a host. 
     
     
         30 . The set according to  claim 23 , wherein the first thermostable DNA polymerase preparation is produced using eukaryotic cells as a host. 
     
     
         31 . The set according to  claim 30 , wherein the second thermostable DNA polymerase is produced using bacterial cell as a host.