Real-time quantitative PCR.
نویسنده
چکیده
Those who have worked in the field of quantitative polymerase chain reaction (PCR) since the early 1990s have accepted many of the tedious aspects of the early assays as routine. Difficulties associated with early quantitative PCR techniques included: (i) ensuring that the PCR was within the linear range of amplification (that portion where the PCR signal is directly proportional to the input copy number) and (ii) finding a suitable method to detect the product once linear amplification was achieved. To ensure linearity, researchers would amplify serial dilutions of cDNA (1) or alter the amplification cycle for each gene (2). Others would add a competitor template (3), perform limiting dilution assays (4), or use a PCR mimic with a similar primer sequence that would be coamplified along with the product (5). Detection of the amplicon generally resorted to running gels and DNA detection using a stain, radioactivity, or probe. For those who have converted to using real-time quantitative PCR, the old days are but a memory. In real-time PCR, worrying about linear amplification, running gels, working with radioactivity, and gathering numbers are a thing of the past. The ability to generate data within a 2to 3-h period is now a reality. Other advantages of real-time PCR include enhanced sensitivity, high throughput, use of a closed-tube system, reduced variation, the ability to simultaneously multiplex reactions, and lack of post-PCR manipulations. The technology to detect PCR products in real time, i.e., during the reaction, has been available for more than 5 years, but has seen a dramatic increase in use over the past 2 years. A Medline search using the key words TaqMan or real-time and PCR yielded 19 citations in 1996, 28 citations in 1997, and 52, 157, and 409 citations in 1998, 1999, and 2000, respectively. At the time of this writing, there were 551 citations in 2001 (Fig. 1). Various manufacturers are touting realtime PCR instrumentation and affiliated technology
منابع مشابه
Evaluation of a new set of Real-Time PCR for Brucella detection within human and animal samples
A quantitative TaqMn Real-Time PCR assay was developed and its diagnostic value on human serum and livestock samples were evaluated. Brucella species could be distributed through communities as a biological agent. Rapid detection of biological threat agents is critical for timely therapeutic administration. Quantitative real-time PCR provides a rapid, sensitive and specific tool for molecular i...
متن کاملمقایسه تاثیر مهاری ژل APF و ژل سدیم فلوراید (NAF) بر روی غلظت میکروارگانیزم های پوسیدگی زا در محیط دهان با روش Quantitative Real Time PCR
Background and purpose: Several factors are involved in caries prevention in children. One of the most effective factors is the appropriate use of fluoride. Fluoride induces its main effects in caries prevention through antibacterial effects and topical contact with enamel. In this study the inhibitory effects of sodium fluoride (NaF) and acidulated phosphate fluoride (APF) gels on cariogenic m...
متن کاملQuantitative detection of chicken meat routine mislabeling in emulsion type sausages and burgers by SYBR green real time PCR assay
ABSTRACT- Today, the authenticity of meat products with less costly and desirable species has increased. Therefore and considering religious, economicalor public health concerns, proper actions should be taken to prevent such frauds. In this study, real time PCR assay was applied for rapid, sensitive and specific identification and quantification of chicken tissue in meat products. Specific pri...
متن کاملDevelopment and Evaluation of Real-Time RT-PCR Test for Quantitative and Qualitative Recognition of Current H9N2 Subtype Avian Influenza Viruses in Iran
Avian influenza H9N2 subtype viruses have had a great impact on Iranian industrial poultry production economy since introduction in the country. To approach Rapid and precise identification of this viruses as control measures in poultry industry, a real time probe base assay was developed to directly detect a specific influenza virus of H9N2 subtype -instead of general detection of Influenza A ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Methods
دوره 25 4 شماره
صفحات -
تاریخ انتشار 2001