Real-time PCR instruments

In order to apply the real-time PCR technology as a rapid, accurate, and direct detection tool for field or point-of-testing applications, it is highly desirable to miniaturize the real-time PCR instrument. To miniaturize the real-time PCR and make it a lab-on-a-chip method, one must realize the following two key functions (1) Control the on-chip thermal cycling, i.e., control the temperature of the PCR reaction wells on the chip. (2) Detect the fluorescent signals during the PCR. 

TaqMan assays are commercially available for many SNPs and can be designed on demand by Applied Biosystems. The method requires a real-time PCR instrument or a fluorescent plate reader to measure the fluorescence of the two dyes at the end of the PCR reaction. 

The absolute quantification assay is used to quantify unknown samples by interpolating their quantity from a standard curve. Absolute quantification might be used to correlate a viral copy number with a disease status. It is of interest to the scientist to know the exact copy number of the target DNA in a given biological sample in order to monitor the progress of the disease. Absolute quantification can be performed with data from aU real-time PCR instruments however, knowledge of the absolute quantities of the standards is an absolute prerequisite in order to run the analysis, and most appropriate by some independent means. 

The instrumentation for real-time PCR includes a thermal cycler with a computer, a spectrophotometer for fluorescence detection, and software for acquisition and analysis of data.105,109... 

To obtain accurate results in qMSP/QM-MSP analyses, the best conditions have to be worked out to achieve reliable standard curves during the test trials. This could be achieved with the use of unmethylated and methylated templates such as placental DNA and in vitro methylated DNA, respectively, and by performing bisulfite treatment as described. To find the best condition for each gene, purify the DNA, calculate the concentration and copy numbers, and dilute the DNA sequentially (e.g., 1,1/2,1/4,1/8, 1/16, 1/32, and 1/64) and perform real-time PCR with several dilutions of the primers (e.g., 25, 50, 75ng each in various combinations). For example, you should see the amplification plots as indicated in Fig. 9.4 with 50% (Fig. 9.4A) or 25% (Fig. 9.4B) sequential dilution. These test trials and any other quantitative PCR (qPCR)ZQM-MSP experiments need to be done in duplicate or triplicate to ensure that the required skills and instruments for equal pipeting are in place, or the impacts could be minimized by averaging the results of the triplicate experiments. Similar to MSP, for each qMSP or QM-MSP trials use placental and... 

Leb V, Stocher M, Valentine-Thon E, Holzl G, Kessler H, Stekel H, et al. Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments. J Clin Microbiol 2004 42 585-90. 

For older or nonoptimaUy collected or stored buccal swabs or decomposed samples, it can happen that a part of the measured DNA comes from bacteria or fiingi. In addition, for those samples the DNA yield expected will be lower than that listed in Table 1.9. Other biological material, such as teeth, bones, or connective tissue, or boiled, grilled, or smoked meat, contains less DNA. In this case a determination of DNA concentration by real-time PCR is necessary. Using a real-time instrument it is possible to determine the total amount of DNA in a sample or the amount of DNA from a specific species. 

DNA from Vertebrates For the differentiation of DNA from bacteria and the DNA from vertebrates, analysis can utilize primers specific for genomic or mitochondrial DNA. Real-time PCR with primers specific for conserved sequences of the mitochondrial cytochrome b gene detects DNA from all mammals and most fishes. For the detection of chondrichtyes (ray, shark) and prawns, other primers have to be chosen. The analysis can be performed with unlabeled primers. Amplicons can be detected by SybrGreen. For most instruments, control samples with known concentrations can be used as markers, and using these the DNA concentration of samples is calculated automatically. 

For some applications or for quality assurance, it can be necessary to control whether instruments, surfaces, reaction tubes, or used buffers are contaminated with DNA, RNA, DNAse, or RNAse. For the detection of DNAse or RNAse, known amounts of DNA or RNA are incubated with the suspected buffers or in the tubes at 37 to 40°C for 30 to 60 min. After this, real-time PCR is performed with the untreated DNA or RNA as control. If the analysis of incubated DNA or RNA shows a higher value than the control, and the internal process control gives no hint of inhibition, DNAse or RNAse was present. For the detection of DNA or RNA contaminations, humid swab samples (in TE or phosphate-buffered saline buffer) have to be collected from surfaces or instruments and the nucleic acids extracted into the TE buffer. The buffer can also be incubated in the suspected reaction tube or pipette tip. Real-time PCR is performed with universal primers specific for cytochrome b, human DNA, or any other DNA/RNA that is identified as a source of contamination in the laboratory. 

Mix/DNA sample to the reaction plates. Perform real-time PCR detection of dotA using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) or comparable instrument. Extrapolate the number of C. burnetii genomes present in the DNA sample from the standard curve. 

MPN-PCR is an easy quantification method, which does not require a standard like in competitive PCR or special instrumentation Hke in real-time PCR. However, the quantification based on MPN-PCR encounter many problems, particularly when appUed to environmental samples. This is due to the uneven distribution of bacteria and their tendency to aggregate, which makes it difficult to obtain homogenous suspensions. In addition, known biases associated with conventional PCR may render quantification based on MPN-PCR unreliable. 

---