浙江预防医学
浙江預防醫學
절강예방의학
ZHEJIANG JOURNAL OF PREVENTIVE MEDICINE
2014年
8期
761-763,767
,共4页
宋启发%叶硕%徐景野%朱国良%章丹阳
宋啟髮%葉碩%徐景野%硃國良%章丹暘
송계발%협석%서경야%주국량%장단양
副溶血性弧菌%热稳定直接溶血素%转录%实时荧光定量 PCR
副溶血性弧菌%熱穩定直接溶血素%轉錄%實時熒光定量 PCR
부용혈성호균%열은정직접용혈소%전록%실시형광정량 PCR
Vibrio parahaemolyticus%Thermostable direct hemolysin%Transcription%Real -time fluorescent quantitative PCR
目的：运用实时荧光定量 PCR 检测24株副溶血性弧菌热稳定直接溶血素（TDH）基因转录水平差异。方法普通 PCR 法检测分离自患者、海产品和环境中的副溶血性弧菌 tdh 基因，tdh 阳性菌株提取细菌总 RNA 后逆转录合成 cDNA，检测无 DNA 污染后定量至同一浓度，采用实时荧光定量 PCR 同时检测 cDNA 产物中 tdh 基因和内标基因16 S rRNA 的 Ct 值，ΔCt 等于 tdh 的 Ct 值减去16 S rRNA 的 Ct 值，以ΔCt 值反应 tdh 相对于内标基因16 S rRNA 的转录水平。结果24株副溶血性弧菌的 tdh Ct 值、内标基因16 S rRNA Ct值和各样本两者之差的ΔCt 值结果范围分别为18.04～25.95、8.30～10.93和8.28～15.34，ΔCt 最大值与最小值差为7.06，最高转录水平菌株为最低菌株的133倍（ΔΔCt ＝27.06）。结论副溶血性弧菌 tdh 基因转录水平存在较大差异，该差异性形成的分子机制以及与该菌的致病性的关系有待进一步研究。
目的：運用實時熒光定量 PCR 檢測24株副溶血性弧菌熱穩定直接溶血素（TDH）基因轉錄水平差異。方法普通 PCR 法檢測分離自患者、海產品和環境中的副溶血性弧菌 tdh 基因，tdh 暘性菌株提取細菌總 RNA 後逆轉錄閤成 cDNA，檢測無 DNA 汙染後定量至同一濃度，採用實時熒光定量 PCR 同時檢測 cDNA 產物中 tdh 基因和內標基因16 S rRNA 的 Ct 值，ΔCt 等于 tdh 的 Ct 值減去16 S rRNA 的 Ct 值，以ΔCt 值反應 tdh 相對于內標基因16 S rRNA 的轉錄水平。結果24株副溶血性弧菌的 tdh Ct 值、內標基因16 S rRNA Ct值和各樣本兩者之差的ΔCt 值結果範圍分彆為18.04～25.95、8.30～10.93和8.28～15.34，ΔCt 最大值與最小值差為7.06，最高轉錄水平菌株為最低菌株的133倍（ΔΔCt ＝27.06）。結論副溶血性弧菌 tdh 基因轉錄水平存在較大差異，該差異性形成的分子機製以及與該菌的緻病性的關繫有待進一步研究。
목적：운용실시형광정량 PCR 검측24주부용혈성호균열은정직접용혈소（TDH）기인전록수평차이。방법보통 PCR 법검측분리자환자、해산품화배경중적부용혈성호균 tdh 기인，tdh 양성균주제취세균총 RNA 후역전록합성 cDNA，검측무 DNA 오염후정량지동일농도，채용실시형광정량 PCR 동시검측 cDNA 산물중 tdh 기인화내표기인16 S rRNA 적 Ct 치，ΔCt 등우 tdh 적 Ct 치감거16 S rRNA 적 Ct 치，이ΔCt 치반응 tdh 상대우내표기인16 S rRNA 적전록수평。결과24주부용혈성호균적 tdh Ct 치、내표기인16 S rRNA Ct치화각양본량자지차적ΔCt 치결과범위분별위18.04～25.95、8.30～10.93화8.28～15.34，ΔCt 최대치여최소치차위7.06，최고전록수평균주위최저균주적133배（ΔΔCt ＝27.06）。결론부용혈성호균 tdh 기인전록수평존재교대차이，해차이성형성적분자궤제이급여해균적치병성적관계유대진일보연구。
Objective To measure the transcription level of thermostable direct hemolysin gene (TDH)in 24 strains of Vibrio parahaemolyticus.Methods Total RNA was extracted from strains of Vibrio parahaemolyticus which were isolated from patients,seafood and environment.The RNA was proved TDH positive with routine PCR method;then the real -time fluorescent quantitative PCR was carried out to obtain the cycle of threshold (Ct)of THD and internal standard of 16s rRNA.Transcription level of THD compared with 16s rRNA was designated as ΔCt which was calculated as Ct value of THD minus Ct value of 16s rRNA.Results Ct values of THD,16s rRNA and the difference between them of the 24 strains was 18.04 ~25.95,8.30 ~10.93 and 8.28 ~15.34 respectively.The difference between the maximum and the minimum of ΔCt was 7.06;the highest transcription level was 133 (ΔΔCt =27.06 )times of the lowest one.Conclusion A great difference of transcription level of THD in Vibrio parahaemolyticus has been proved and further study is needed to clarify the possible molecular mechanisms and relationship between the transcription level of THD and pathogenic mechanism.
