JP2013055888A - Quick and easy bacterium detection from blood culture sample - Google Patents
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本発明は、血液培養サンプルからの迅速な微生物検出、同定に関する。 The present invention relates to rapid microbial detection and identification from blood culture samples.
敗血症は重篤な全身感染症で、確定診断には血液中の起因微生物の検出・同定が必須である。
近年、癌治療や臓器移植など医療の高度化に伴い、敗血症発症のリスクの高い重症患者が増えている。
また、院内感染の観点からメチシリン耐性黄色ブドウ球菌（ＭＲＳＡ）をはじめとする多剤耐性菌が敗血症の起因菌となることも多く、適切な抗菌薬を選択し患者を救命するためには、血液中の起因菌を可能な限り迅速に検出・同定することが臨床上重要である。
Sepsis is a serious systemic infection, and detection and identification of the causative microorganisms in the blood is essential for a definitive diagnosis.
In recent years, with the advancement of medical treatment such as cancer treatment and organ transplantation, the number of severe patients with a high risk of developing sepsis is increasing.
In addition, from the viewpoint of nosocomial infection, multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are often the causative bacteria for sepsis. It is clinically important to detect and identify the causative bacteria as quickly as possible.
これらの検出を行うに際して、遺伝子検査は非常に迅速に検出、同定が行われるメリットがある。一方、遺伝子検査では、検査に投入できる血液量が少ないことから、一般に、感度を上げる等の目的で測定前に血液培養が行われている。
血液培養液を試料とした従来の遺伝子検査では、血液培養液中の微生物菌体から核酸を抽出し遺伝子検査に供する方法、あるいは血液培養液中の微生物菌体を回収して溶菌させてから遺伝子検査に供する方法が主に用いられてきた。（非特許文献１、非特許文献２）
これらの方法には、核酸抽出工程あるいは溶菌工程に要する手間と時間により検査の迅速性が失われるという欠点がある。さらに核酸抽出工程あるいは溶菌工程を失敗すれば続く遺伝子検査を適切に行えないという問題点も指摘されている。
When performing these detections, genetic testing has the advantage of being detected and identified very quickly. On the other hand, in gene testing, since the amount of blood that can be input into the test is small, blood culture is generally performed before measurement for the purpose of increasing sensitivity.
In a conventional genetic test using a blood culture solution as a sample, nucleic acid is extracted from microbial cells in the blood culture solution and used for genetic testing, or the microbial cells in the blood culture solution are collected and lysed before the gene. Methods used for inspection have been mainly used. (Non-patent document 1, Non-patent document 2)
These methods have the disadvantage that the rapidity of the test is lost due to the labor and time required for the nucleic acid extraction step or the lysis step. Further, it has been pointed out that if the nucleic acid extraction process or the lysis process fails, the subsequent genetic test cannot be performed properly.
本発明は、上記の遺伝子検査に関する問題点を解決しようとするものである。 The present invention seeks to solve the problems associated with the genetic testing described above.
本発明者らは上記課題に鑑み鋭意検討した結果、血液培養液の濃度をある一定の範囲にすることにより、従来技術よりも迅速かつ正確にＭＲＳＡを検出できることを見出し、本発明を完成させるに至った。
すなわち、本発明は以下のような構成からなる。
［項１］
血液培養ボトル中の微生物または微生物由来の核酸を検出する方法であって以下の（１）〜（３）の工程を含む方法。
（１）血液培養ボトルの液層の一部または全部を試料として採取する工程
（２）（１）で採取した試料を、続く（３）の工程での終濃度が、血液培養液に対して希釈倍率２倍を越え１００倍以下となるように水またはトリス緩衝液で希釈する工程
（３）（２）の試料を遺伝子検査に供して、試料に含まれる微生物由来の核酸を検出する工程
［項２］
血液培養ボトル中の微生物または微生物由来の核酸を検出する方法であって以下の（１）〜（３）の工程を含む方法。
（１）血液培養ボトルの液層の一部または全部を試料として採取する工程
（２）（１）で採取した試料を水またはトリス緩衝液で希釈し、次いで、得られた希釈試料にカオトロピック塩を含む溶液を添加し、カオトロピック塩を含む溶液を添加した試料からシリカを含むカラムによって核酸を精製する工程であって、続く（３）の工程での終濃度が、血液培養液に対して希釈倍率２倍を越え１００倍以下となるようにする工程
（３）（２）の試料を遺伝子検査に供して、試料に含まれる微生物由来の核酸を検出する工程
As a result of intensive studies in view of the above problems, the present inventors have found that MRSA can be detected more quickly and more accurately than the prior art by setting the concentration of the blood culture solution within a certain range, and the present invention is completed. It came.
That is, the present invention has the following configuration.
[Claim 1]
A method for detecting a microorganism or a nucleic acid derived from a microorganism in a blood culture bottle, comprising the following steps (1) to (3):
(1) Step of collecting a part or all of the liquid layer of the blood culture bottle as a sample (2) The sample collected in (1) is subjected to the final concentration in the subsequent step (3) with respect to the blood culture solution. The step of (3) diluting with water or Tris buffer so that the dilution ratio is more than 2 times and not more than 100 times, the sample of (2) is subjected to genetic testing, and the nucleic acid derived from microorganisms contained in the sample is detected [ Item 2]
A method for detecting a microorganism or a nucleic acid derived from a microorganism in a blood culture bottle, comprising the following steps (1) to (3):
(1) Step of collecting part or all of the liquid layer of a blood culture bottle as a sample (2) The sample collected in (1) is diluted with water or Tris buffer, and then the obtained diluted sample is chaotropic salt A nucleic acid is purified by a column containing silica from a sample to which a solution containing a chaotropic salt is added, and the final concentration in the subsequent step (3) is diluted with respect to the blood culture solution. The step of (3) and (2) in which the magnification is doubled to 100 but not larger than 100, and the sample of the sample is subjected to a genetic test to detect a nucleic acid derived from a microorganism contained in the sample.
本発明によれば、血液培養によって増殖させた微生物の検出を迅速かつ簡便に行うことが可能になる。 According to the present invention, it is possible to quickly and easily detect microorganisms grown by blood culture.
本発明の実施形態の一局面は、血液培養ボトル中の微生物または微生物由来の核酸を検出する方法であって、以下の（１）〜（３）の工程を含む方法である。
（１）血液培養ボトルの液層の一部または全部を試料として採取する工程
（２）（１）で採取した試料を、続く（３）の工程での終濃度が、血液培養液に対して希釈倍率２倍を越え１００倍以下となるように水またはトリス緩衝液で希釈する工程
（３）（２）の試料を遺伝子検査に供して、試料に含まれる微生物由来の核酸を検出する工程
One aspect of the embodiment of the present invention is a method for detecting a microorganism or a nucleic acid derived from a microorganism in a blood culture bottle, and includes the following steps (1) to (3).
(1) Step of collecting a part or all of the liquid layer of the blood culture bottle as a sample (2) The sample collected in (1) is subjected to the final concentration in the subsequent step (3) with respect to the blood culture solution. Step of diluting with water or Tris buffer so that the dilution ratio exceeds 2 times and not more than 100 times (3) A step of subjecting the sample of (2) to genetic testing and detecting a nucleic acid derived from a microorganism contained in the sample
通常、試料の希釈は、検出対象である微生物菌体の濃度を低下させるため、検査にはふさわしくないと考えられてきた。
しかし意外なことに、発明者らの検討によれば、血液培養液をそのまま核酸増幅に供しても遺伝子が検出できないのに対し、核酸増幅系における血液培養液の終濃度が５％未満になるよう希釈を行えば、微生物の溶菌操作等を行うことなく簡易な操作で核酸増幅および検出が可能であることが明らかになった。
In general, dilution of a sample has been considered to be unsuitable for testing because it reduces the concentration of microbial cells to be detected.
Surprisingly, however, according to the study by the inventors, the gene cannot be detected even if the blood culture solution is directly subjected to nucleic acid amplification, whereas the final concentration of the blood culture solution in the nucleic acid amplification system is less than 5%. Thus, it was revealed that nucleic acid amplification and detection can be performed with simple operations without performing lysis of microorganisms.
本発明における遺伝子検査とは、核酸を検出する方法および／または核酸から微生物を同定する方法である。検査法としては従来公知の各方法を用いることができ特に限定されないが、好ましくは（１）核酸増幅反応により核酸を増幅する工程（２）該増幅産物を検出する工程、の二つの工程を含む方法である。 The genetic test in the present invention is a method for detecting a nucleic acid and / or a method for identifying a microorganism from a nucleic acid. Conventionally known methods can be used as the inspection method and are not particularly limited, but preferably include two steps of (1) a step of amplifying a nucleic acid by a nucleic acid amplification reaction and (2) a step of detecting the amplification product. Is the method.
核酸の増幅工程に用いられる具体的な核酸増幅方法は特に限定されず、適宜公知の方法を用いることができる。例えば、ＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）法、ＮＡＳＢＡ（Ｎｕｃｌｅｉｃ ａｃｉｄ ｓｅｑｕｅｎｃｅ ｂａｓｅｄ ａｍｐｌｉｆｉｃａｔｉｏｎ）法、ＴＭＡ（Ｔｒａｎｓｃｒｉｐｔｉｏｎ−ｍｅｄｉａｔｅｄ ａｍｐｌｉｆｉｃａｔｉｏｎ）法、ＳＤＡ（Ｓｔｒａｎｄ Ｄｉｓｐｌａｃｅｍｅｎｔ Ａｍｐｌｉｆｉｃａｔｉｏｎ）法等があげられるが、ＰＣＲ法を用いることが好ましい。なお、これらの各方法において、増幅反応の条件は特に制限されず、従来公知の方法により行うことができる。 The specific nucleic acid amplification method used in the nucleic acid amplification step is not particularly limited, and a known method can be used as appropriate. For example, a PCR (Polymerase Chain Reaction) method, a NASBA (Nucleic acid sequence based amplification) method, a TMA (Transscription-mediated amplification) method, a SDA (Strand Displacement Amplification method), and a SDA (Strand Displacement Amplification method) are preferred. In each of these methods, the conditions for the amplification reaction are not particularly limited, and can be performed by a conventionally known method.
核酸増幅産物の検出工程に用いられる具体的な核酸検出方法としては特に限定されず、適宜公知の方法を用いることができる。例えば、アガロースゲル電気泳動法、シークエンス法、ＤＮＡプローブ法、リアルタイムＰＣＲ法等があげられるが、ＤＮＡプローブ法を用いることが好ましい。 The specific nucleic acid detection method used in the detection step of the nucleic acid amplification product is not particularly limited, and a known method can be used as appropriate. For example, an agarose gel electrophoresis method, a sequencing method, a DNA probe method, a real-time PCR method and the like can be mentioned, and it is preferable to use a DNA probe method.
上記検出工程の前に、希釈して得られた試料にカオトロピック塩を含む溶液を添加し、カオトロピック塩を含む溶液を添加した試料からシリカを含むカラムによって核酸を精製する工程を含んでも良い。
この工程に用いる溶液やカラムは特に限定されない。カオトロピック塩としてはグアニジンイソチオシアネート、グアニジン塩酸塩等が例示され、好ましくは２〜４ｍｏｌ／Ｌのグアニジン塩酸塩である。また、カラムとしては、シリカモノリスが例示され、市販のものを用いることができる。
Before the detection step, a step of adding a solution containing a chaotropic salt to a sample obtained by dilution and purifying a nucleic acid from a sample to which a solution containing a chaotropic salt is added using a column containing silica may be included.
The solution and column used in this step are not particularly limited. Examples of chaotropic salts include guanidine isothiocyanate and guanidine hydrochloride, and preferably 2 to 4 mol / L guanidine hydrochloride. Moreover, as a column, a silica monolith is illustrated and a commercially available thing can be used.
本発明の方法の検出対象となる微生物、または、本発明の方法の検出対象である核酸の由来となる微生物は、血液培養ボトルによって培養可能な微生物であれば特に限定されない。 The microorganism to be detected by the method of the present invention or the microorganism from which the nucleic acid to be detected by the method of the present invention is derived is not particularly limited as long as it is a microorganism that can be cultured in a blood culture bottle.
本発明の方法において、血液培養方法は特に限定されない。例えば市販の血液培養ボトルを用いて定法により培養すればよい。
血液培養に用いる培地も特に限定されず、市販の血液培養ボトルを用いることができる。
In the method of the present invention, the blood culture method is not particularly limited. For example, it may be cultured by a conventional method using a commercially available blood culture bottle.
The medium used for blood culture is not particularly limited, and a commercially available blood culture bottle can be used.
本発明において、血液培養液の希釈に用いる溶液は、遺伝子検査を著しく阻害するものでなければ特に限定されない。希釈に用いる溶液としては、水、トリス緩衝液が挙げられる。 In the present invention, the solution used for dilution of the blood culture solution is not particularly limited as long as it does not significantly inhibit the genetic test. Examples of the solution used for dilution include water and Tris buffer.
本発明において、遺伝子検査を行うために適切な血液培養液の希釈倍率は、血液培養液に対して２倍を越え１００倍以下である。これは、検出液中の終濃度で０．１％以上５％未満に相当する。検出系中の血液培養液の終濃度が４％以下である（希釈倍率２５倍を超える）ことがより好ましく、３．４％以下である（希釈倍率３０倍を超える）ことがより好ましく、３％以下である（希釈倍率３３．３倍を超える）ことがより好ましい。終濃度の下限については特に限定されないが、一般的に検出系中の試料濃度が小さくなるほど検出されなくなる可能性が高まる（測定系の検出限界にもよるが）ため、十分な検出能を確保するという観点から０．１％以上であることが好ましい。 In the present invention, the appropriate dilution ratio of the blood culture solution for performing the genetic test is more than 2 times and 100 times or less of the blood culture solution. This corresponds to a final concentration in the detection solution of 0.1% or more and less than 5%. The final concentration of the blood culture medium in the detection system is more preferably 4% or less (more than 25 times the dilution factor), more preferably 3.4% or less (more than 30 times the dilution factor). % (More than 33.3 times dilution ratio) is more preferable. The lower limit of the final concentration is not particularly limited. In general, the lower the sample concentration in the detection system, the higher the possibility that it will not be detected (depending on the detection limit of the measurement system). In view of the above, it is preferably 0.1% or more.
試料を希釈する工程には、得られた希釈試料にカオトロピック塩を含む溶液を添加し、カオトロピック塩を含む溶液を添加した試料からシリカを含むカラムによって核酸を精製する工程を含んでも良い。この場合、希釈倍率は、全体の工程を通して、続く核酸検出工程での終濃度が、血液培養液に対して希釈倍率２倍を越え１００倍以下となるようにすればよい。
なお、シリカを含むカラムによって核酸を精製する工程における希釈倍率は、カラムに供した血液培養液の液量と、カラムから溶出させた溶出液の液量との比率で決定する。
The step of diluting the sample may include a step of adding a solution containing a chaotropic salt to the obtained diluted sample and purifying the nucleic acid by a column containing silica from the sample to which the solution containing the chaotropic salt is added. In this case, the dilution rate may be such that the final concentration in the subsequent nucleic acid detection step is over 2 times the dilution rate with respect to the blood culture solution and 100 times or less throughout the entire process.
In addition, the dilution rate in the step of purifying nucleic acid using a column containing silica is determined by the ratio between the volume of the blood culture solution supplied to the column and the volume of the eluate eluted from the column.
以下に実施例を示して本発明を具体的に説明するが、本発明は実施例に限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
〔実施例１：血液培養ボトルで培養したコアグラーゼ陰性ブドウ球菌の直接検出〕
（１）試料の調製
メチシリン耐性コアグラーゼ陰性ブドウ球菌を含む血液培養液を１０ｍＭのＴｒｉｓ−ＨＣｌ（ｐＨ７．５）で検出系１０μＬでの終濃度が１／１０、１／２０、１／５０、１／１００、１／２００、１／３００、１／４００、１／５００、１／１０００の濃度になるようそれぞれ希釈し、試料とした。陰性コントロール（ＮＣ）として水を使用した。
以下に血液培養液の検出系中の試料終濃度と試料希釈倍率との関係を表１に示す。
[Example 1: Direct detection of coagulase-negative staphylococci cultured in blood culture bottles]
(1) Preparation of sample A blood culture solution containing methicillin-resistant coagulase-negative staphylococci with 10 mM Tris-HCl (pH 7.5) at a final concentration of 1/10, 1/20, 1/50, 1 Samples were prepared by diluting to concentrations of / 100, 1/200, 1/300, 1/400, 1/500, and 1/1000, respectively. Water was used as a negative control (NC).
Table 1 shows the relationship between the final sample concentration in the blood culture medium detection system and the sample dilution rate.
（２）核酸増幅および融解曲線解析
上記試料および陰性コントロールにそれぞれ下記試薬を添加して、ＰＣＲ法および蛍光プローブ法を利用してメチシリン耐性遺伝子を検出した。核酸増幅および融解曲線は下記条件で実施した。核酸増幅および融解曲線解析には東洋紡績社製ＧＥＮＥＣＵＢＥ（登録商標）を使用した。
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the sample and the negative control, respectively, and the methicillin resistance gene was detected using the PCR method and the fluorescent probe method. Nucleic acid amplification and melting curves were performed under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis.
試薬
以下の試薬を含む１０μＬ溶液を調製した。
核酸プライマー（配列番号１） ３００ｎＭ
核酸プライマー（配列番号２） １５００ｎＭ
核酸プローブ（配列番号３、３’末端をＢＯＤＩＰＹ−ＦＬ標識） ３００ｎＭ
×１０緩衝液 １μＬ
ｄＮＴＰ ０．２ｍＭ
ＭｇＳＯ４ ４ｍＭ
ＢＳＡ １μｇ
ＤＭＳＯ ０．７５μＬ
ＫＯＤ ｐｌｕｓ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（東洋紡績製） ０．３Ｕ
試料 １μＬ
Reagents A 10 μL solution containing the following reagents was prepared.
Nucleic acid primer (SEQ ID NO: 1) 300 nM
Nucleic acid primer (SEQ ID NO: 2) 1500 nM
Nucleic acid probe (SEQ ID NO: 3, 3 ′ end labeled with BODIPY-FL) 300 nM
× 10 buffer 1μL
dNTP 0.2 mM
MgSO4 4 mM
1 μg of BSA
DMSO 0.75μL
KOD plus DNA polymerase (manufactured by Toyobo) 0.3U
Sample 1μL
核酸増幅および融解曲線解析
９４℃・２分
（以上１サイクル）
９７℃・１秒
６０℃・３秒
６３℃・５秒
（以上５０サイクル）
９４℃・３０秒
３９℃・３０秒
３９℃〜７５℃（０．０９℃／秒で温度上昇）
Nucleic acid amplification and melting curve analysis 94 ° C, 2 minutes (1 cycle above)
97 ° C · 1 second 60 ° C · 3 seconds 63 ° C · 5 seconds (over 50 cycles)
94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rise at 0.09 ° C / second)
結果
図１は、温度上昇にともなう蛍光強度の変化を、グラフの横軸を温度、縦軸を蛍光シグナルの微分値として解析結果を表した図である。血液培養液を終濃度１／１０または１／２０となるように希釈した試料からはメチシリン耐性遺伝子を検出することができなかった。これに対して、終濃度を１／５０以下となるように希釈した試料からはメチシリン耐性遺伝子が検出された。
以上の結果から、検出系に血液培養液成分が少なくとも５％以上含まれる場合は、血液培養液中の菌に対する遺伝子検査が阻害されることが示された。さらに、本実施例により検出系に含まれる血液培養液成分が２％以下であれば遺伝子検査が実施可能であることが明らかになった。
Results FIG. 1 is a graph showing the results of analysis of changes in fluorescence intensity with increasing temperature, with the horizontal axis of the graph representing temperature and the vertical axis representing the differential value of the fluorescence signal. A methicillin resistant gene could not be detected from a sample obtained by diluting the blood culture solution to a final concentration of 1/10 or 1/20. In contrast, a methicillin resistant gene was detected from a sample diluted to a final concentration of 1/50 or less.
From the above results, it was shown that when the detection system contains at least 5% or more of a blood culture medium component, genetic testing for bacteria in the blood culture medium is inhibited. Furthermore, the present example revealed that genetic testing can be carried out if the blood culture fluid component contained in the detection system is 2% or less.
〔実施例２：血液培養ボトルで培養したコアグラーゼ陰性ブドウ球菌のシリカモノリスによる核酸精製からの検出〕
（１）試料の調製
メチシリン耐性コアグラーゼ陰性ブドウ球菌を含む血液培養液を溶液量３００μＬ中の終濃度が１、１／１０、１／４０となるように精製水で希釈した。これをモノリス構造のシリカ固層カラムを含むチップ（以下モノリスチップ、ＧＬサイエンス製）を用いて核酸精製を行い、精製した核酸を含む溶液を試料とした。陰性コントロール（ＮＣ）として水を使用した。
以下に血液培養液から精製した核酸を含む溶液試料の検出系中の試料終濃度と試料希釈倍率との関係を表２に示す。なお、検出系での終濃度とは血液培養液からの精製物が核酸増幅系に占める割合によって示される。
[Example 2: Detection of nucleic acid purification by silica monoliths of coagulase-negative staphylococci cultured in blood culture bottles]
(1) Preparation of Sample A blood culture solution containing methicillin-resistant coagulase-negative staphylococci was diluted with purified water so that the final concentration in the solution volume of 300 μL was 1, 1/10, 1/40. This was subjected to nucleic acid purification using a chip (hereinafter, monolith chip, manufactured by GL Science) containing a monolithic silica solid layer column, and a solution containing the purified nucleic acid was used as a sample. Water was used as a negative control (NC).
Table 2 shows the relationship between the sample final concentration in the detection system of the solution sample containing nucleic acid purified from the blood culture medium and the sample dilution rate. The final concentration in the detection system is indicated by the ratio of the purified product from the blood culture solution to the nucleic acid amplification system.
（２）核酸精製
核酸精製は以下の手順で行った。血液培養液を精製水で希釈した以降の工程は全てＧＥＮＥＣＵＢＥ（東洋紡）を使用して行った。
メチシリン耐性コアグラーゼ陰性ブドウ球菌を含む血液培養液を（１）に記載の終濃度となるように精製水で希釈した。各希釈液量は３００μＬとなるように調製した。この希釈液３００μＬにＧＥＮＥＣＵＢＥ用溶解吸着液（東洋紡）５５０μＬを添加し、６５℃で５分間加熱した。加熱後の溶液をモノリスチップに通した。次に、ＧＥＮＥＣＵＢＥ用洗浄液（東洋紡）３５０μＬをモノリスチップに通し、モノリスチップを洗浄した。この洗浄工程は２回行った。洗浄後、１０ｍＭ水酸化カリウム２０μＬでモノリスチップに吸着していた核酸を溶出し回収した。この回収した溶液を遺伝子検査試料とした。
（３）核酸増幅および融解曲線解析
上記試料および陰性コントロールにそれぞれ下記試薬を添加して、ＰＣＲ法および蛍光プローブ法を利用してメチシリン耐性遺伝子を検出した。核酸増幅および融解曲線は下記条件で実施した。核酸増幅および融解曲線解析には東洋紡績社製ＧＥＮＥＣＵＢＥ（登録商標）を使用した。
(2) Nucleic acid purification Nucleic acid purification was performed according to the following procedure. All steps after diluting the blood culture with purified water were performed using GENECUBE (Toyobo).
A blood culture solution containing methicillin-resistant coagulase-negative staphylococci was diluted with purified water to the final concentration described in (1). Each dilution was prepared to be 300 μL. 550 μL of GENECUBE dissolution adsorbent (Toyobo) was added to 300 μL of this diluted solution, and heated at 65 ° C. for 5 minutes. The heated solution was passed through a monolith chip. Next, 350 μL of GENECUBE cleaning liquid (Toyobo) was passed through the monolith chip to wash the monolith chip. This washing step was performed twice. After washing, the nucleic acid adsorbed on the monolith chip was eluted and recovered with 20 μL of 10 mM potassium hydroxide. This collected solution was used as a genetic test sample.
(3) Nucleic acid amplification and melting curve analysis The following reagents were added to the sample and the negative control, respectively, and the methicillin resistance gene was detected using the PCR method and the fluorescent probe method. Nucleic acid amplification and melting curves were performed under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis.
核酸増幅および融解曲線解析試薬
以下の試薬を含む１３．２μＬ溶液を調製した。
核酸プライマー（配列番号１） ３００ｎＭ
核酸プライマー（配列番号２） １５００ｎＭ
核酸プローブ（配列番号３、３’末端をＢＯＤＩＰＹ−ＦＬ標識） ３００ｎＭ
×１０緩衝液 １．３μＬ
ｄＮＴＰ ０．２ｍＭ
ＭｇＳＯ４ ４ｍＭ
ＢＳＡ １．３μｇ
ＤＭＳＯ ０．９８μＬ
ＫＯＤ ｐｌｕｓ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（東洋紡績製） ０．４Ｕ
試料 ４μＬ
Nucleic Acid Amplification and Melting Curve Analysis Reagent A 13.2 μL solution containing the following reagents was prepared.
Nucleic acid primer (SEQ ID NO: 1) 300 nM
Nucleic acid primer (SEQ ID NO: 2) 1500 nM
Nucleic acid probe (SEQ ID NO: 3, 3 ′ end labeled with BODIPY-FL) 300 nM
× 10 buffer 1.3μL
dNTP 0.2 mM
MgSO4 4 mM
BSA 1.3μg
DMSO 0.98μL
KOD plus DNA polymerase (manufactured by Toyobo) 0.4U
Sample 4μL
核酸増幅および融解曲線解析
９４℃・２分
（以上１サイクル）
９７℃・１秒
６０℃・３秒
６３℃・５秒
（以上５０サイクル）
９４℃・３０秒
３９℃・３０秒
３９℃〜７５℃（０．０９℃／秒で温度上昇）
Nucleic acid amplification and melting curve analysis 94 ° C, 2 minutes (1 cycle above)
97 ° C · 1 second 60 ° C · 3 seconds 63 ° C · 5 seconds (over 50 cycles)
94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rise at 0.09 ° C / second)
結果
本実施例の結果を表３に示す。血液培養液を希釈せずに核酸精製を行った後、精製された核酸を含む溶液を試料として遺伝子検査を行ったところ、該試料からメチシリン耐性遺伝子は検出されなかった（表では結果を「×」で表示、「ＮＣ」はネガティブコントロール）。これに対して、血液培養液の割合が１／１０または１／４０となるように精製水で希釈し、該希釈液から核酸を精製し、精製された核酸を含む溶液を試料として遺伝子検査を行うと、該試料からメチシリン耐性遺伝子が検出された（表では結果を「○」で表示）。
この結果は二つのことを示唆している。すなわち、第一にはシリカ固層カラムによる核酸精製では、血液培養液に含まれる遺伝子検査の阻害要因を完全には除去できないことである。第二には、シリカ固層カラムで血液培養液からの核酸精製を行う場合でも、血液培養液を予め希釈することが遺伝子検査を正確に実施する上で非常に有効な手段となることである。
Results The results of this example are shown in Table 3. After nucleic acid purification without diluting the blood culture solution, genetic testing was performed using a solution containing the purified nucleic acid as a sample. As a result, no methicillin-resistant gene was detected from the sample (the results are shown as “×” in the table). "," NC "is a negative control). On the other hand, it is diluted with purified water so that the ratio of the blood culture solution becomes 1/10 or 1/40, nucleic acid is purified from the diluted solution, and a gene test is performed using a solution containing the purified nucleic acid as a sample. When this was done, a methicillin resistance gene was detected from the sample (results are indicated by “◯” in the table).
This result suggests two things. That is, firstly, nucleic acid purification using a silica solid-phase column cannot completely remove an inhibitory factor for genetic testing contained in a blood culture solution. Second, even when nucleic acid purification is performed from a blood culture medium using a silica solid layer column, diluting the blood culture medium in advance is a very effective means for accurately performing genetic testing. .
〔比較例１：リン酸緩衝液（ｐＨ７．５）で希釈した血液培養液からのコアグラーゼ陰性ブドウ球菌の直接検出〕
（１）試料の調製
メチシリン耐性コアグラーゼ陰性ブドウ球菌を含む血液培養液を１０ｍＭのリン酸カリウム‐ナトリウム緩衝液（ｐＨ７．５）で検出系１０μＬでの終濃度が１／１０、１／５０、１／１００、１／２００、１／５００、１／１０００の濃度になるようそれぞれ希釈し、試料とした。陰性コントロール（ＮＣ）として水を使用した。
[Comparative Example 1: Direct detection of coagulase-negative staphylococci from blood cultures diluted with phosphate buffer (pH 7.5)]
(1) Preparation of sample A blood culture solution containing methicillin-resistant coagulase-negative staphylococci with 10 mM potassium phosphate-sodium buffer (pH 7.5) at a final concentration of 1/10, 1/50, 1 Samples were prepared by diluting to concentrations of / 100, 1/200, 1/500, and 1/1000, respectively. Water was used as a negative control (NC).
（２）核酸増幅および融解曲線解析
上記試料および陰性コントロールにそれぞれ下記試薬を添加して、ＰＣＲ法および蛍光プローブ法を利用してメチシリン耐性遺伝子を検出した。核酸増幅および融解曲線は下記条件で実施した。核酸増幅および融解曲線解析には東洋紡績社製ＧＥＮＥＣＵＢＥ（登録商標）を使用した。
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the sample and the negative control, respectively, and the methicillin resistance gene was detected using the PCR method and the fluorescent probe method. Nucleic acid amplification and melting curves were performed under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis.
試薬
以下の試薬を含む１０μＬ溶液を調製した。
核酸プライマー（配列番号１） ３００ｎＭ
核酸プライマー（配列番号２） １５００ｎＭ
核酸プローブ（配列番号３、３’末端をＢＯＤＩＰＹ−ＦＬ標識） ３００ｎＭ
×１０緩衝液 １μＬ
ｄＮＴＰ ０．２ｍＭ
ＭｇＳＯ４ ４ｍＭ
ＢＳＡ １μｇ
ＤＭＳＯ ０．７５μＬ
ＫＯＤ ｐｌｕｓ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（東洋紡績製） ０．３Ｕ
試料 １μＬ
Reagents A 10 μL solution containing the following reagents was prepared.
Nucleic acid primer (SEQ ID NO: 1) 300 nM
Nucleic acid primer (SEQ ID NO: 2) 1500 nM
Nucleic acid probe (SEQ ID NO: 3, 3 ′ end labeled with BODIPY-FL) 300 nM
× 10 buffer 1μL
dNTP 0.2 mM
MgSO4 4 mM
1 μg of BSA
DMSO 0.75μL
KOD plus DNA polymerase (manufactured by Toyobo) 0.3U
Sample 1μL
核酸増幅および融解曲線解析
９４℃・２分
（以上１サイクル）
９７℃・１秒
６０℃・３秒
６３℃・５秒
（以上５０サイクル）
９４℃・３０秒
３９℃・３０秒
３９℃〜７５℃（０．０９℃／秒で温度上昇）
Nucleic acid amplification and melting curve analysis 94 ° C, 2 minutes (1 cycle above)
97 ° C · 1 second 60 ° C · 3 seconds 63 ° C · 5 seconds (over 50 cycles)
94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rise at 0.09 ° C / second)
結果
図２は、温度上昇にともなう蛍光強度の変化を、グラフの横軸を温度、縦軸を蛍光シグナルの微分値として解析結果を表した図である。本比較例では終濃度を１／２００となるように希釈した試料のみメチシリン耐性遺伝子が検出された。
実施例１の結果と比較すると、血液培養液中のコアグラーゼ陰性ブドウ球菌由来メチシリン耐性遺伝子を検出することができる血液培養液終濃度の範囲が著しく狭まっていることが明らかである。これはリン酸緩衝液によって核酸増幅が阻害されていることを示唆している。
本比較例の結果は、血液培養液の希釈に使用する溶液としては、リン酸緩衝液は不適当であり、トリス緩衝液の方がより好ましいことを示している。
Results FIG. 2 is a diagram showing the analysis results of changes in fluorescence intensity with temperature rise, with the horizontal axis of the graph being temperature and the vertical axis being the differential value of the fluorescence signal. In this comparative example, the methicillin resistance gene was detected only in the sample diluted to a final concentration of 1/200.
Compared with the results of Example 1, it is clear that the range of the final concentration of blood culture solution in which the coagulase-negative staphylococcal methicillin resistance gene in the blood culture solution can be detected is significantly narrowed. This suggests that nucleic acid amplification is inhibited by the phosphate buffer.
The result of this comparative example shows that the phosphate buffer is inappropriate as the solution used for diluting the blood culture solution, and the Tris buffer is more preferable.
本発明により、従来技術よりも迅速かつ正確に血液培養ボトルで生育させた微生物を検出することができ、診断や医療の分野に貢献する。 According to the present invention, microorganisms grown in a blood culture bottle can be detected more quickly and more accurately than in the prior art, contributing to the fields of diagnosis and medicine.
Claims (2)
（１）血液培養ボトルの液層の一部または全部を試料として採取する工程
（２）（１）で採取した試料を、続く（３）の工程での終濃度が、血液培養液に対して希釈倍率２倍を越え１００倍以下となるように水またはトリス緩衝液で希釈する工程
（３）（２）の試料を遺伝子検査に供して、試料に含まれる微生物由来の核酸を検出する工程 A method for detecting a microorganism or a nucleic acid derived from a microorganism in a blood culture bottle, comprising the following steps (1) to (3):
(1) Step of collecting a part or all of the liquid layer of the blood culture bottle as a sample (2) The sample collected in (1) is subjected to the final concentration in the subsequent step (3) with respect to the blood culture solution. Step of diluting with water or Tris buffer so that the dilution ratio exceeds 2 times and not more than 100 times (3) A step of subjecting the sample of (2) to genetic testing and detecting a nucleic acid derived from a microorganism contained in the sample
（１）血液培養ボトルの液層の一部または全部を試料として採取する工程
（２）（１）で採取した試料を水またはトリス緩衝液で希釈し、次いで、得られた希釈試料にカオトロピック塩を含む溶液を添加し、カオトロピック塩を含む溶液を添加した試料からシリカを含むカラムによって核酸を精製する工程であって、続く（３）の工程での終濃度が、血液培養液に対して希釈倍率２倍を越え１００倍以下となるようにする工程
（３）（２）の試料を遺伝子検査に供して、試料に含まれる微生物由来の核酸を検出する工程 A method for detecting a microorganism or a nucleic acid derived from a microorganism in a blood culture bottle, comprising the following steps (1) to (3):
(1) Step of collecting part or all of the liquid layer of a blood culture bottle as a sample (2) The sample collected in (1) is diluted with water or Tris buffer, and then the obtained diluted sample is chaotropic salt A nucleic acid is purified by a column containing silica from a sample to which a solution containing a chaotropic salt is added, and the final concentration in the subsequent step (3) is diluted with respect to the blood culture solution. The step of (3) and (2) in which the magnification is doubled to 100 but not larger than 100, and the sample of the sample is subjected to a genetic test to detect a nucleic acid derived from a microorganism contained in the sample.
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| JP2002537824A (en) * | 1999-03-02 | 2002-11-12 | キングス カレッジ ロンドン | Bacterial identification |
| JP2007502618A (en) * | 2003-08-15 | 2007-02-15 | ユニバーシティー オブ サウス フロリダ | Materials and methods for capturing pathogens from samples and removing aurintricarboxylic acid |
| JP2009537167A (en) * | 2006-05-19 | 2009-10-29 | ジーンオーム サイエンシズ インコーポレイテッド | Method for extracting deoxyribonucleic acid (DNA) from microorganisms that may be present in a blood sample |
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| JP2002537824A (en) * | 1999-03-02 | 2002-11-12 | キングス カレッジ ロンドン | Bacterial identification |
| JP2007502618A (en) * | 2003-08-15 | 2007-02-15 | ユニバーシティー オブ サウス フロリダ | Materials and methods for capturing pathogens from samples and removing aurintricarboxylic acid |
| JP2009537167A (en) * | 2006-05-19 | 2009-10-29 | ジーンオーム サイエンシズ インコーポレイテッド | Method for extracting deoxyribonucleic acid (DNA) from microorganisms that may be present in a blood sample |
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