JP2023093129A - 細菌の検出方法および細菌の検出用キット - Google Patents
細菌の検出方法および細菌の検出用キット Download PDFInfo
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Abstract
Description
細菌の核酸を増幅および検出する技術には、様々な方法が用いられている。例えば、PCRを利用するPCR法の他、転写-逆転写協奏反応(TRC)法、転写媒介性増幅(TMA)法、拡散配列ベース増幅(NASBA)法、ループ媒介性等温増幅(LAMP)法、スマート増幅プロセス(SMAP)法、等温キメラプライマー開始核酸増幅(ICAN)法等が知られている。
本発明の一検出方法は、分解工程と、失活工程と、検出工程とを含む。
分解工程は、細菌を含み得る生体試料と、タンパク質分解酵素を含むPCR緩衝液とを混合した検体混合液について、タンパク質分解酵素の反応を進行させる工程である。
失活工程は、タンパク質分解酵素による反応後、検体混合液に含まれるタンパク質分解酵素を失活させる工程である。タンパク質分解酵素を失活させる方法は特に限定されないが、例えば、検体混合液を加熱する、タンパク質分解酵素の阻害剤を検体混合液に添加する、タンパク質分解酵素を自己消化させる等の方法が挙げられる。なかでも、加熱による失活が、簡便かつ確実にタンパク質分解酵素を失活させる観点から好ましい。
検出工程は、失活工程後の検体混合液の少なくとも一部を、PCR反応組成物に添加してPCR産物を増幅し、当該PCR産物を検出する工程である。
本発明の一実施形態に係る細菌の検出用キット(以下、「本発明の一キット」と称する場合がある)は、タンパク質分解酵素を含むPCR緩衝液と、PCR反応組成物とを備える。本発明の一キットが備えるPCR反応組成物は、眼科性細菌感染症の起炎病原体の少なくとも1種を検出するためのPCRプライマー対を含む。また、本発明の一キットは、上述のコントロール用PCR反応組成物をさらに含んでいることが好ましい。本発明の一キットが備える各構成については、上述の〔本発明の一検出方法〕の説明を援用可能である。
(1-1.PCR産物の増幅確認)
本発明の一検出方法に用いるPCRプライマー対および蛍光標識プローブを用いたPCR産物の増幅実験を行った。本実験に用いたPCRプライマー対を以下に示す:
GAPDH遺伝子検出用
(フォワード)5'-tgtgctcccactcctgatttc-3'(配列番号1)
(リバース)5'-cctagtcccagggctttgatt-3'(配列番号2)
TBP遺伝子検出用
(フォワード)5'-gcaccactccactgtatccc-3'(配列番号3)
(リバース)5'-cccagaactctccgaagctg-3'(配列番号4)
エンテロコッカス検出用
(フォワード)5'-agcctcggaattgagaatga-3'(配列番号5)
(リバース)5'-gaccttagctggtggtctgg-3'(配列番号6)
クレブシエラ検出用
(フォワード)5'-aaccaggcgtcgataat-3'(配列番号7)
(リバース)5'-gtttacggcgcaatcc-3'(配列番号8)
B群溶血性レンサ球菌検出用
(フォワード)5'-ctctagtggctggtgcattg-3'(配列番号9)
(リバース)5'-ccatttgctgggcttgatta-3'(配列番号10)
A群溶血性レンサ球菌検出用
(フォワード)5'-aggcggacatgcctttgtta-3'(配列番号11)
(リバース)5'-tgcctacaacagcactttgg-3'(配列番号12)
肺炎レンサ球菌検出用
(フォワード)5'-cacgcaatctagcagatgaagca-3'(配列番号13)
(リバース)5'-tcgtgcgttttaattccagct-3'(配列番号14)
緑膿菌検出用
(フォワード)5'-gccgaggtcatggaattc-3'(配列番号15)
(リバース)5'-atccgcgccatcatcttc-3'(配列番号16)
表皮ブドウ球菌検出用
(フォワード)5'-ccagaacgtgaYtctgacaaa-3'(配列番号17)
(リバース)5'-tcaccRactttgatttgacca-3'(配列番号18)
大腸菌検出用
(フォワード)5'-catgccgcgtgtatgaagaa-3'(配列番号19)
(リバース)5'-cgggtaacgtcaatgagcaaa-3'(配列番号20)
黄色ブドウ球菌検出用
(フォワード)5'-catcggaaacattgtgttctgtatg-3'(配列番号21)
(リバース)5'-tttggctggaaaatataactctcgta-3'(配列番号22)
ノカルディア検出用
(フォワード)5'-ccggaaacctgcagagatgt-3'(配列番号23)
(リバース)5'-tacgcgctggcaacataaga-3'(配列番号24)
vanA遺伝子検出用
(フォワード)5'-ggatagctactcccgccttt-3'(配列番号25)
(リバース)5'-cagcctgctcaattaagattttg-3'(配列番号26)
mecA遺伝子検出用
(フォワード)5'-cctctgctcaacaagttcca-3'(配列番号27)
(リバース)5'-aacgttgtaaccaccccaag-3'(配列番号28)
gyrA遺伝子変異検出用
(フォワード)5'-gaacaaggtatgacgcccgata-3'(配列番号29)
(リバース)5'-ggccattcttaccattgcttca-3'(配列番号30)。
GAPDH遺伝子検出用
5'-aaaagagctaggaaggacaggcaacttggc-3'(配列番号31)(FAM標識)
TBP遺伝子検出用
5'-acccccatcactcctgccacgc-3'(配列番号32)(ROX標識)
エンテロコッカス検出用
5'-cccatctcgggttaccgaattcaga-3'(配列番号33)(FAM標識)
クレブシエラ検出用
5'-acaggaaagacaagactatgcagacc-3'(配列番号34)(ROX標識)
B群溶血性レンサ球菌検出用
5'-catgctgatcaagtgacaactccaca-3'(配列番号35)(FAM標識)
A群溶血性レンサ球菌検出用
5'-tggtggcggcgcaggcggcttcaac-3'(配列番号36)(ROX標識)
肺炎レンサ球菌検出用
5'-ctccctgtatcaagcgttttcggc-3'(配列番号37)(FAM標識)
緑膿菌検出用
5'-cgacaaccgcaaggaagccga-3'(配列番号38)(ROX標識)
表皮ブドウ球菌検出用
5'-ccattcatgatgccagttgaggacg-3'(配列番号39)(FAM標識)
大腸菌検出用
5'-tattaactttactcccttcctccccgctgaa-3'(配列番号40)(ROX標識)
黄色ブドウ球菌検出用
5'-aagccgtcttgataatctttagtagtaccgaagctggt-3'(配列番号41)(FAM標識)
ノカルディア検出用
5'-agtcccgcaacgagcgcaaccctt-3'(配列番号42)(ROX標識)
vanA遺伝子検出用
5'-tcctgtttttgttaagccggcgc-3'(配列番号43)(FAM標識)
mecA遺伝子検出用
5'-aatcgatggtaaaggttggcaaaaaga-3'(配列番号44)(ROX標識)
gyrA遺伝子変異検出用
野生型(Cアリル)5'-ttgaagaatcacc-3'(配列番号45)(FAM標識)
変異型(Tアリル)5'-tgaaaaatcaccatga-3'(配列番号46)(ALEXA594標識)
変異型(Aアリル)5'-tgaataatcaccatga-3'(配列番号47)(ALEXA594標識)。
次に、各標的核酸を含む試験用プラスミドが個別の状態、または全て混合された状態で存在する検体混合液を調製した。各標的核酸を含む試験用プラスミドのコピー数は、反応ごとに50コピーとした。その他の条件は前記(1-1.PCR産物の増幅確認)と同様にして、本発明の一検出方法を実施した。結果を以下表2に示す。表2では、結果としてCq値を示している。
眼科性感染症が疑われる患者から得られた生体試料を検体として、細菌由来の核酸の検出実験を行った。検体以外の条件は前記(1-1.PCR産物の増幅確認)と同様にして、本発明の一検出方法を実施した。ただし、本実験のチューブFでは、ノカルディア検出用のPCRプライマー対および蛍光標識プローブを含まないPCR反応固体組成物を用いた。結果を以下表3に示す。表3では、結果としてCq値を示している。
Claims (8)
- 細菌を含み得る生体試料と、タンパク質分解酵素を含むPCR緩衝液とを混合した検体混合液について、前記タンパク質分解酵素の反応を進行させる分解工程と、
前記検体混合液に含まれる前記タンパク質分解酵素を失活させる失活工程と、
前記失活工程後の前記検体混合液の少なくとも一部をPCR反応組成物に添加してPCR産物を増幅し、当該PCR産物を検出する検出工程と、を含む細菌の検出方法。 - 前記生体試料は、眼球または眼球付属物の少なくとも一部を含む眼科検体である、請求項1に記載の細菌の検出方法。
- 前記PCR反応組成物は、眼科性細菌感染症の起炎病原体の少なくとも1種を検出するためのPCRプライマー対を含む、請求項1または2に記載の細菌の検出方法。
- 前記起炎病原体は、エンテロコッカス属、クレブシエラ属、ノカルディア属、レンサ球菌属、スタフィロコッカス属、緑膿菌および大腸菌からなる群より選ばれる少なくとも1種である、請求項3に記載の細菌の検出方法。
- 前記PCR反応組成物は、メチシリン耐性遺伝子、バンコマイシン耐性遺伝子およびキノロン耐性遺伝子の少なくとも何れかを検出するためのPCRプライマー対を含む、請求項1から3の何れか1項に記載の細菌の検出方法。
- 前記PCR反応組成物は、DNAジャイレース遺伝子およびトポイソメラーゼIV遺伝子の少なくとも何れかにおける、キノロン耐性を示す遺伝子変異の有無を検出するためのPCRプライマー対を含む、請求項1から5の何れか1項に記載の細菌の検出方法。
- 前記PCR反応組成物は、メチシリン耐性遺伝子、バンコマイシン耐性遺伝子およびキノロン耐性遺伝子の少なくとも何れかを検出するためのPCRプライマー対と、前記キノロン耐性を示す遺伝子変異の有無を検出するためのPCRプライマー対と、を含む、請求項6に記載の細菌の検出方法。
- タンパク質分解酵素を含むPCR緩衝液と、PCR反応組成物とを備え、
前記PCR反応組成物は、細菌感染症の起炎病原体の少なくとも1種を検出するためのPCRプライマー対を含む、細菌の検出用キット。
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