Real Time PCR and its Application in Plant Pathology-III

Relevant Features of Real-Time PCR

           Rapidity:

Compared with classical PCR, real-time PCR is rapid to provide reliable data. Typically, the time of a whole real-time PCR run ranges from 20 min to 2 hour. In fact, the time needed to shift temperature is a major limiting factor responsible for the duration of a classical PCR experiment. The Light Cycler™ PCR machine uses capillaries instead of tubes, which are heated by light instead of a heating block. As a result, the time necessary to heat the PCR mixture is considerably reduced (from 15 second to 1–2 second). In addition, recording the amplification in real-time avoids collecting samples at different steps of the PCR experiment, making the process less tedious and time-consuming (Gachon et al., 2004).

Sensitivity:

Diagnostic sensitivity is defined as a measure of the degree to detect the target pathogen in the sample, which may result in false negative responses. It relates to the lowest number of pathogens reliably detected per assay or sample (Lopeze et al., 2003). Too low sensitivity often leads to false negatives. Thus, a high degree of diagnostic accuracy is characterized by the ability to detect, true and precisely the target micro organism from a sample without interference from non target components. The high degree of sensitivity of molecular methods made pre-symptomatic detection and quantification of pathogens possible. Real-time PCR provides a high sensitivity for the detection of DNA or RNA due to a combination of the amplification performed by the PCR step and the system of detection (Baustin, 2002). It is therefore a very convenient technique for studies with a limited amount of starting material or for assessing the expression of a high number of genes from minute quantities of RNA (Bago et al., 2002). The detection is based on the measurement of the fluorescence emitted by probes incorporated into the newly formed PCR product, or alternatively released into the buffer during the amplification of the PCR product.

Specificity:

Diagnostic specificity is defined as a measure of the degree to which the method is affected by non target components present in a sample, which may result in false positive responses. It’s the capability to detect the organism of interest in the absence of false positives and negatives (Lopeze et al., 2003). In a study carried out on four pea thioredoxin h (TRXh) encoding genes, Montrichard et al. (2003) noticed that real-time PCR yielded weaker signals than expected from northern blot analyses. This observation was explained by a crosshybridization of the probe to the RNA encoding another isoform during the northern blot procedure. Indeed, in contrast to techniques requiring the hybridization of nucleic acids several hundred base pairs long, such as cDNA-based microarray and northern blotting, short oligonucleotide-mediated real-time PCR guarantees a high specificity in the detection of the target sequence. In fact, specificity is achieved by the use of two target sequence-specific oligonucleotides, and this can be enhanced by increasing the number of oligonucleotides nested within the initial amplification product. In this respect, FRET-mediated probes seem to ensure a higher specificity than SYBR green® (Shimada et al., 2003). In any case, specificity of the process can be checked after completion of the PCR run, by testing the nature of the amplified product with gel electrophoresis, melting curves, and sequencing data.

Quantification:

Nucleic acid quantification meant the addition of radio-labeled deoxythymidine triphosphate (dTTP) to cell cultures or one of many possible in vitro experimental preparations and measuring their incorporation into nucleic acids by TCA (trichoroacetic acid) precipitation. Although radioactive incorporation is a quantitative technique and gave the investigator an idea of the global changes in the nucleic acid population of their experimental system, it was not satisfactory for identifying or quantifying specific genes or transcripts (Dorak, 2006). Real time PCR-based analyses combine ‘traditional’ end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in ‘real time’ during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. Basically, real-time quantitative PCR may be used for quantifying DNA or RNA abundance, leading to two major types of applications: foreign DNA detection and quantification, and gene expression studies (Gachon et al., 2004).

Application of Real Time PCR in Plant Disease Diagnostics

a. Identification and Diagnosis of Plant Disease:

Accurate identification and early detection of pathogens is a crucial step in plant disease management (Schaad and Frederick, 2002, Schaad et al., 2003). Development of real time-PCR plays a substantial role in this regard. From the middle of the 1990s a wide range of PCR assays were developed and applied to the diagnosis of infectious diseases, as well to the improved detection of pathogens. Currently, real-time PCR is considered the gold standard method for detection of plant pathogens, as it allows high sensitivity and specificity in the detection of one or several pathogens in a single assay (Lopeze et al., 2003). Various real-time PCR assays including TaqMan, Molecular Beacons, Primer-Probe Energy Scorpion Primers, dual probe systems such as SYBR R Green; showed high sensitivity and specificity for the detection plant pathogenic microorganisms.

             b. Quantification of pathogenic microorganisms associated with plants

Quantification of a pathogen is an important aspect because it can be used to estimate its potential risk regarding disease development, establishment and spread of pathogen and economic loss. Quantification is essential mainly for soil-borne pathogens although it is well established that quantity of initial inoculum has a critical importance in the subsequent epidemic caused by pathogens disseminated by the wind (Paplomatas, 2006). Recently the process of quantifying target DNA has been simplified considerably with the advent of real-time PCR. This method avoids the usual need for post-reaction processing, as the amplified products are detected by a built-in fluorimeter as they accumulate. This is done by using non-specific DNA binding dyes (e.g. SYBR Green) or fluorescent probes that are specific to the target DNA (Wittwer et al., 1997). The principle underlying real-time PCR is that the larger the amount of target DNA present in the sample being tested, the quicker the reaction progresses and enters the exponential phase of amplification. The amount of PCR amplicon produced at each cycle is measured, using the fluorescent dyes or probes, and for each sample tested the cycle threshold (Ct) is calculated. This is the cycle number at which a statistically significant increase in fluorescence is detected. The Ct increases with decreasing amounts of target DNA. A calibration curve relating Ct to known amounts of target DNA is constructed and used to quantify the amount of initial target DNA in an unknown sample. Software supplied with real-time PCR machines is used to rapidly analyze the results (Ward et al., 2004). The fluorescence intensity emitted during this process reflects the amplicons concentration in real time. The real time data will serve as useful basis for establishing inoculum threshold levels and detailed analysis of disease epidemics (Ward et al., 2004).

c. Detection of Multiplexing

Crops can be infected by numerous pathogens and they may be present in plants in complexes. Therefore, it is desirable to develop technology that can detect multiple pathogens simultaneously. The methodological limitations however, are in many cases the reasons for developing simplex or assays only including few targets. Multiplex PCR, a PCR variant which is designed to amplify multiple targets by using multiple primer sets in the same reaction, has been applied in many tests. Multiplex PCR assays can be tedious and time consuming to establish requiring lengthy optimization processes (Elnifro et al., 2000). Among the drawbacks of such variant PCR assays are that the sensitivity is decreased enormously and the number of different targets to be amplified in one assay is limited (Bamaga et al., 2003). Moreover, the dynamic range of the target present in the sample to be tested is not always reflected in the outcome of the test. That is targets that are present in very low amounts will most of the time not amplified in contrast to those that are present abundantly.

The real-time PCR offer better multiplexing possibilities, however, multiplexing is still limited by the availability of dyes emitting fluorescence at different wavelengths. Thus, detection of more than few pathogens is currently not possible using these systems. The unlimited capability for simultaneous detection of pathogens makes real-time multiplex PCR to be an approach with a potential capacity of detecting all relevant pathogens of a specific crop. In plant pathology the method was applied for identifying nematode bacterial and fungal DNA from pure cultures.

d. Monitoring of Fungicide Resistance

Frequent application of fungicides with a single mode of action incurs a high risk of selecting resistance genotype of plant pathogens. To determine level of resistance to fungicides, the most common traditional technique is direct-planting single spore isolates in media amended with various concentrations of fungicide and determining inhibition of growth or/and spore germination. The entire test can take 1 to 3 weeks longer if the time required to isolate the pathogen from infected plant tissue is included.

With the advancement of molecular techniques particularly the real-time PCR techniques resistance development by pathogen to a particular fungicide can be detected easily within a short period of time. Michailides et al., (2005) indicated that resistance of Alternaria to azoxystrobin and Benzimidazoles resistance in M. fructincola in stone fruit and M. laxa isolates in almonds can be easily detected with allele specific real-time PCR.

Conclusion

Real-time PCR has a remarkable potential in quan­tifying low disease levels with high sensitivity and speed that was inconceivable in plant pathology a few years ago. The technique is extremely promising in order to quantify pathogen populations, whereas other PCR-based techniques qualify only for the identification/detection of the microbial commu­nities. With accurate optimization, real-time PCR can provide specific, reliable, and high throughput detection and quantification of target DNA in vari­ous environmental samples in real time, which is not achievable with other PCR-based methods. In fact, real-time PCR is an ideal technique to measure levels of inoculums threshold, which has a positive impact on epidemiological studies, and for evaluat­ing the efficacy of methodologies used to prevent distribution of the pathogens into non-infected ag­ricultural fields. As knowledge regarding individual microorganisms’ genomes increases, the use of this technique for a broad range of microorganisms will undoubtedly increase. In addition, this growing list of applications suggests that real-time PCR will be an increasingly preferred method in the future, opening new research opportunities associated with a comprehensive understanding of ecology and popu­lation dynamics of pathogens with the final intent of optimizing plant disease management strategies.