Technical Considerations For RT-qPCR Implementation In Food Microbiology....#food #microbiology
What is RT- qPCR?
RT-PCR (Real Time Polymerase Chain Reaction) is the method of quantification of nucleic acids such as DNA and RNA. The method involves the nucleic acid amplification by the help of polymerase chain reaction in real time i. e. not at its end.
Why RT-qPCR in food microbiology?
The method is nowadays being used in food microbiology in order to identify, detect, and quantify the pathogens or beneficial flora such as probiotics or fermenting microbes. ISO standards also provide guidelines to detect food-borne pathogens qualitatively by PCR.
1. Quality of nucleic acids extract.
Nucleic acid extraction is the initial step in the analysis process. Sample quality is perhaps the most important component to preserve the biological meaning and ensure reproducibility of the analysis. Nowadays, it is easy to get high quality and quantity of DNA yields. Most procedures use commercial extraction kits, used sometimes with some adaptations depending on the food matrix, to ensure satisfactory results. By contrast with DNA, intact RNA extraction is much more laborious and sensitive, especially from complex or fatty food matrices. Some extraction methods which are compatible with subsequent RT-qPCR, have also been developed for various foods. Due to high sensitivity, RNA should be analyzed quickly. Nowadays automated capillary-electrophoresis equipment is the most appropriate to know the sample quality. An RNA integrity number (RIN) can be calculated to check suitability of samples for RT-qPCR analysis. Upstream steps of the detection procedure like sampling and sample preparation are often overlooked as compared to analytical part, in spite of the technical breakthroughs.
2. Detection chemistry
In food microbiology, mostly two detection chemistries are used.
· DNA binding dye assay using SYBR green
· Hydrolysis probe method using TaqMan probe
Due to non specificity of SYBR green assay, it can be used for different gene assays. It is inexpensive, flexible and gives accurate results providing validation of specificity by melt curve or dissociation curve. TaqMan chemistry is more expensive but highly specific that the presence of certain hydrolysis probes makes sure the presence of an amplicon. Furthermore, multiplexing reactions are also possible; nevertheless, an important optimization phase is required for their setup.
3. Quantiﬁcation methods
Accurate quantification is very important for food microbiology applications. For absolute quantification, a comparison is generated of Cq values with a standard curve, which has been generated by amplification of unknown amounts of target genes. The method requires analogous amplification efficiencies of all standards and samples. Hence the standard curve template must be chosen with great care. Gene expression is estimated through relative quantification. Quantification results are indicated as target/reference ratio. Relative quantification is simpler as compared to absolute quantification. This is because it does not necessitate the use of reliable standard in every PCR. However, it is applicable only to the sample run within same PCR.
Amplification efficiencies must be considered while performing relative quantification. This is because many PCR don’t display ideal efficiency. Amplification efficiency values must be calculated and reported for each amplicon, specifically when Cq values comparison is to be made between different samples from different food matrix, or if different strains are quantified.
4. One or two step RT-qPCR
RT-qPCR can be performed in single step within a single tube or in two steps with performing reverse transcription separately from qPCR. One step protocols have less chances of DNA contamination and ultimately less risk of experimental variation. On the other hand, the risk of RNA degradation elevates when if a long term analysis is performed. Is such cases, two step protocols are followed.
5. Experimental variations of RT-qPCR
Almost all the steps are error prone. Due to its high sensitivity small variations can result in non negligible errors in results. Intra-assay variability can be measured by performing RT-qPCR in triplicate. To measure inter-assay variability a reference sample is included in each experiment. Variability because of biological factors is of prime importance in food microbiology. Hence, biological triplicate is recommended with the target RNA, cDNA or DNA on other replicates.
6. Controls and normalization
Multiple controls should be added to evaluate RT efficiency, template contamination with DNA, and variation in master mixture composition. An internal amplification control (IAC) is required in order to know about the food components that inhibit PCR. Cq shifts ≥2 or ≥3 between qPCR having IAC along with sample and solely IAC were proposed as cutoffs. For diagnostic purpose qPCR, Cq delay ≥1 is considered as cutoff inhibition value because partial inhibition can also decrease the lower quantification limit.
7. Mode of expression of RT-qPCR data
Bacterial population’s absolute quantification is expressed as genome equivalent/ml or CFU number/ml. expression of results in GE/ml requires the information of total genome weight and copy number of target gene. Some variations in the results of CFU/ml and GE/ml can be observed as in case of incomplete lysis or in the presence of bacterial chains. For expressional analysis, we take the ratio of normalized expressional level of experimental to control sample for relative quantification. Mostly, log transformed results are obtained for final gene expression, which makes the data more symmetrical to apply for statistical parametric tests.