استفاده از روش PCR رقابتي و Real-time PCR مطلق به منظور كمي سنجي جمعيت باكتري توليد كننده بوتيرات: بوتيريويبريو فيبري سالونس

Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

سویه¬های باكتری بوتیریویبریو فیبری¬سالونس به عنوان عمده¬ترین گروه باكتری¬های تولید كننده بوتیرات در دستگاه گوارش بسیاری از حیوانات و همچنین انسان شناخته شده¬اند. در این مطالعه دو تكنیك مبتنی بر DNA شامل PCR رقابتی و Real-time PCR به منظور شمارش باكتری¬های گونه بوتیریویبریو فیبری¬سالونس استفاده شدند. در ابتدا آغازگرهای اختصاصی برای منطقه 16S rDNA باكتری بوتیریویبریو فیبری¬سالونس به منظور تكثیر یك قطعه 213 جفت بازی استفاده شدند. یك قطعه رقابتگر با تفاوت طول 50 جفت باز بیشتر ساخته شد و در ناقل پلاسمیدی pTZ57R/T همسانه¬سازی شد. كمیت رقابتگر پلاسمیدی با استفاده از نانودراپ اسپكتروفتومتری سنجیده شد و به صورت سریالی رقیق سازی شد. رقت¬های حاصل به همراه DNA استخراج شده از مایع شكمبه بطور همزمان تكثیر شدند. به منظور كمی¬سازی محصولات PCR پس از عكس برداری از ژل آگارز و استفاده از نرم افزار ImageJ ، مقادیر تكثیر شده از DNA هدف در مقابل مقادیر تكثیر شده از رقابتگر به صورت لگاریتمی ترسیم شدند. از ضریب تبیین (R2) به عنوان معیاری جهت ارزیابی دقت تكنیك استفاده گردید. برای توسعه Real-time PCR، قطعه 213 جفت بازی تكثیر شده و همسانه‌سازی شده در ناقل پلاسمیدی pTZ57R/T به منظور رسم منحنی استاندارد استفاده شد. نتایج نشان دادند كه هر دو روش قابلیت استفاده برای شمارش باكتری¬های جنس بوتیریویبریو فیبری¬سالونس را دارا هستند و بنابراین می¬توانند به عنوان ابزارهای مناسب در تحقیقات مرتبط با این باكتری مورد استفاده قرار بگیرند.

Introduction Butyrivibrio fibrisolvens strains are presently recognized as the major butyrate-producing bacteria found in the rumen and digestive track of many animals and also in the human gut. In this study we reported the development of two DNA based techniques, quantitative competitive (QC) PCR and absolute based Real-Time PCR, for enumerating Butyrivibrio fibrisolvens strains. Despite the recent introduction of real-time PCR method for the rapid quantification of the target DNA sequences, use of quantitative competitive PCR (QC-PCR) technique continues to play an important role in nucleic acid quantification since it is more cost effective. The procedure relies on the co-amplification of the sequence of interest with a serially diluted synthetic DNA fragment of the known concentration (competitor), using the single set primers. A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR. Materials and Methods At first reported species-specific primers targeting the 16S rDNA region of the bacterium Butyrivibrio fibrisolvens were used for amplifying a 213 bp fragment. A DNA competitor differing by 50 bp in length from the 213 bp fragment was constructed and cloned into pTZ57R/T vector. The competitor was quantified by NanoDrop spectrophotometer and serially diluted and co-amplified by PCR with total extracted DNA from rumen fluid samples. PCR products were quantified by photographing agarose gels and analyzed with Image J software and the amount of amplified target DNA was log plotted against the amount of amplified competitor. Coefficient of determination (R2) was used as a criterion of methodology precision. For developing the Real-time PCR technique, the 213 bp fragment was amplified and cloned into pTZ57R/T was used to draw a standard curve. Results and Discussion The specific primers of Butyrivibrio fibrisolvens were successfully used for amplifying the specific fragments from this bacteria. The main and important factors for increasing the accuracy of Q-C PCR is the degree of similarity between competitor and target fragment. In this study the competitor fragment was highest homology to target sequences. In this regards it seems obtained results have considerable accuracy. The intensity of bands was evaluated and analyzed using Image J software. The results of band intensity analysis showed linear trend between competitor and target in different serial dilution of competitors. The specific fragment from 16S rDNA region of Butyrivibrio fibriolvents Bacteria was amplified using specific primers and cloned in pTZ57R/T plasmid. After amplifying the competitor fragment and target sequence simultaneously, the two bands were detectable in gel electrophoresis. The range of 10-1 to 10-6 serial dilution from competitor was selected for QC-PCR reaction. The results of this section showed that the considerable linear correlation was exist between competitor and target fragment in QC-PCR reaction (R2=0.985). In this study, Real-time PCR was also used for quantification of Butyrivibrio fibriolvents Bacteria strain. Melting analysis was showed that the reaction in Real-time PCR had appropriate condition for amplifying the target sequence. We used a standard in this study. This standard was designed as a vector contain of competitor fragment. This kind of standard was successfully used for QC-PCR and Real-time PCR analyses. Standard competitor could be used for absolute quantification for this bacterial strain. Overall, our results showed that the designed standard and optimized QC-PCR have considerable potential for Butyrivibrio fibriolvents Bacteria strain. Conclusion In this study, the two methods of quantification of nucleotide acids in biological samples were optimized. These two methods were performed in order to optimize Butyrivibrio fibriolvents Bacteria strain quantification. Our results showed that both techniques have the capabilities to use as valuable research methodologies for enumerating the Butyrivibrio fibrisolvens strains.