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.

 

Real Time PCR and its Application in Plant Pathology-II

  Protocol for Real-Time PCR

RT-PCR can be carried out by the one step RT-PCR protocol or the two step RT-PCR protocol. Use only intact, high quality RNA for the best results.

A. One step RT-PCR protocol: one step RT-PCR protocol take mRNA targets (up to 6 kb) and subjects them to reverse transcription and then PCR amplification in a single test tube.

1. Select a one step RT-PCR kit, which should include a mix with the reverse transcriptase and the PCR system such as Taq DNA Polymerase and a proofreading polymerase
2. Obtain all necessary materials, equipment and instruments
3. Prepare a reaction mix, which will include dNTPs, primers, template RNA, necessary enzymes and a buffer solution
4. Add the mix to a PCR tube for each reaction. Then add the template RNA
5. Place PCR tubes in the thermal cycler to begin cycling. The first cycle is reverse transcription to synthesize cDNA. The second cycle is initial denaturation. During this cycle reverse transcriptase is inactivated. The next 40-50 cycles are the amplification program, which consists of three steps: denaturation, annealing and elongation
6. The RT-PCR products can then be analyzed with gel electrophoresis

B. Two step RT-PCR protocol: two steps RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This method is more sensitive than the one step method. 

Step 1:

1. Combine template RNA, primer, dNTP mix, and nuclease free water in a PCR tube
2. Add RNase inhibitor and reverse transcriptase to the PCR tube
3. Place PCR tube in thermal cycler for one cycle and then inactivating reverse transcriptase
4. Proceed directly to PCR or store on ice until PCR can be performed

Step 2:

1. Add a master mix (containing buffer, dNTP mix, MgCl_2, Taq polymerase and nuclease free water) to each PCR tube
2. Add appropriate primer
3. Place PCR tubes in thermal cycler for 30 cycles of the amplification program
4. The RT-PCR products can then be analyzed with gel electrophoresis

                                                                              Continued........

Real Time PCR and its Application in Plant Pathology-I

 Introduction

Diseases in plants cause major production and economic losses in agriculture worldwide. Monitoring of health and detection of diseases in plants is critical for sustainable agriculture (Sankaran et al., 2010). Detection of pathogens in plants showing symptoms can be relatively simple provided one has extensive experience with disease diagnosis and isolation of plant pathogens. On the other hand, detection of pathogens in seeds or asymptomatic propagative materials, such as woody cuttings or potato tubers, can be extremely difficult since few propagules of the pathogen are present. Because of this, sensitive techniques capable of detection of very low numbers of pathogen propagules are needed. Until serological techniques were developed, the only reliable methods available for identification of fungi and bacteria were isolation in culture and performing pathogenicity tests. Serological techniques allowed rapid presumptive diagnosis of bacterial diseases (Hampton et al. 1990). However, the only reliable diagnostic technique for viruses was host indexing. Not until DNA-based techniques were developed could pathogens be detected reliably in asymptomatic plant materials. DNA dot-blotting techniques using specific hybridization probes became very useful by offering very high specificity. However, like serological techniques, hybridization assays using DNA probes were not always very sensitive, and they were very time consuming and required additional technical skills.

Recent advances in biotechnology and molecular biology tools have played a substantial role in the development of rapid, specific and sensitive diagnostic tests (Henson and French 1993; Makkouk and Kumari, 2006). Molecular diagnostic began to develop a real momentum after the invention of polymerase chain reaction (PCR) in the mid 1980s and the first PCR based detection of a pathogen in diseased plants was published in the beginning of 1990s. With the advances in molecular biology and biosystematics, the techniques available have evolved significantly in the last decade, and besides conventional PCR other technologically advanced methodologies such as the second generation PCR known as the real-time PCR and microarrays which allows unlimited multiplexing capability have the potential to bring pathogen detection to a new and improved level of efficiency and reliability (Mumford et al., 2006).

Molecular techniques based on different types of PCR amplification and especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture.

Principle of Real-Time PCR

Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction is a PCR based laboratory technique, which is used to amplify and simultaneously quantify a targeted DNA molecule. Real-Time PCR follows the general principle of polymerase chain reaction except that the progress of the reaction is monitored by a camera or detector in real-time. This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. To monitor the progress of PCR, fluorescent marker are used which binds to the DNA. Hence, as the number of gene copies increases during the reaction so the fluorescence increases. This is advantageous because the efficiency and rate of the reaction can be seen. There is also no need to run the PCR product out on a gel after the reaction (Paplomatas, 2006; Capote et al., 2012).

A probe (Example TaqMan) is designed to anneal to the target sequence between the traditional forward and reverse primers. The probe is labeled at the 5’ end with a reporter fluorochrome and a quencher fluorochrome added at the 3’ end. The probe is designed to have a higher Tm than the primers, and during the extension phase, the probe must be 100% hybridized for success of the assay. As long as fluorochromes are on the probe, the quencher molecule stops all fluorescence by the reporter. However, as Taq polymerase extends the primer, the intrinsic 5’ to 3’ nuclease activity of Taq degrades the probe, releasing the reporter fluorochrome. The amount of fluorescence released during the amplification cycle is proportional to the amount of product generated in each cycle (Makkouk and Kumari, 2006). The final aim, which is the quantification of the unknown DNA, comes from the fact that the initial amount of DNA in the sample is related to the number of cycles needed for the fluorescence to reach a specific cycle threshold (the Ct value), defined as that cycle number at which a statistically significant increase in fluorescence is detected. After generating a calibration curve by plotting Ct against known amounts of template DNA, target DNA can be quantified (Paplomatas, 2006).\

                                                    Real-time PCR methods

Generally, Real-time PCR chemistry can be group into amplicon sequence non-specific method which based on the use of doubled-stranded DNA binding dyes, such as SYBR Green, and sequence specific methods which based on fluorescent labeled probes such as TaqMan, Molecular Beacons or Scorpions (Mackay et al., 2002; Schena et al., 2004; Capote et al., 2012). All of these methods are based upon the hybridization of fluorescently labeled oligonucleotide probe sequences to a specific region within the target amplicon that is amplified using traditional forward and reverse PCR primers.

1. Amplicon sequence non-specific methods

Included in this group are detection methods based on the use of dyes that emit fluorescent light when intercalated into double-stranded DNA (dsDNA). The most utilized fluorescence intercalating dye is SYBR Green I as it has high affinity for double-stranded DNA. This dye binds to the major groove of double-stranded DNA but not single-stranded DNA and so as amplicons accumulate during the PCR process SYBR green binds the double-stranded DNA proportionally and fluorescence emission of the dye can be detected following excitation. Thus the accumulation of DNA amplicons can be followed in real time during the reaction run. At the end of the reaction, products of different length and/or sequence can be observed as distinct fluorescent peaks by heating the reaction from 30–40°C to 95°C whilst continuously monitoring the fluorescence (melting curve analysis). In fact, plotting the first negative derivative of fluorescence vs. temperature, the point at which dsDNA melts will be observed as a drop (peak) in fluorescence. If a single peak representing the specific products is observed, SYBR Green I is a simple and reliable low-cost method for monitoring PCR amplicons and for quantifying template DNA. 

2.Amplicon sequence specific methods

There are several amplicon sequence specific detection methods based on the use of oligonucleotide probes labelled with a donor fluorophore and an acceptor dye (quencher). This includes TaqMan, Molecular beacons, and Scorpion PCR. The probes used to monitor DNA amplification in a PCR are cleaved in the reaction (TaqMan) or undergo a conformation change in the presence of a complementary DNA target (Molecular beacons and Scorpion- PCR) that separate fluorophore and quencher. In TaqMan, the fluorophore is quenched by FRET, whereas in Molecular beacons and Scorpion-PCR, fluorescence quenching is proximal, due to the close contact of fluorophore and quencher (Schena et al., 2004).

2.1.The TaqMan probe method

Fluorescent probes are segments of DNA complimentary to gene of interest that are labeled with a fluorescent dye. The simplest and most commonly used type of probe is the TaqMan-type probe. The TaqMan probe method utilizes a fluorescently labeled probe that hybridizes to an additional conserved region that lies within the target amplicon sequence. These probes are labeled with a fluorescent reporter molecule at one end and a quencher molecule at the other. Hence under normal circumstances the fluorescent emission from the probe is low. However during the PCR the probe binds to the gene of interest and becomes cleaved by the polymerase. Hence the reporter and quencher are physically separated and the fluorescence increases (Schaad et al., 2003; Capote et al., 2012). Fluorescence can be measured throughout the PCR, providing “real-time” analysis of the reaction kinetics and allowing quantification of specific DNA targets. Interpretation of the fluorometric data can be presented during the PCR assay and also facilitates quantification of the amount of sample DNA present in the reaction by ascertaining when (i.e. during which PCR cycle) fluorescence in a given reaction tube exceeds that of a threshold value (Threshold Cycle (Ct)). Comparison between reaction tubes and/or known standards allows quantification of the amount of DNA template present in a given tube (Weller et al., 2006).

2.2.     Molecular beacons

Molecular beacons are fluorescent oligonucleotide probes that are designed to include stem-loop folding. They are complementary nucleotide sequences to the target amplicon. A fluorescent chromophore is attached at the 5′ end of the probe and a quencher molecule is attached at the 3′ end. A stem structure is formed by annealing of the complementary arm sequences that are added on both sides of the probe sequence. When a stem structure is formed, the fluorophore transfers energy to the quencher, and no fluorescence is emitted. However, when the probe hybridizes to the target amplicon during PCR amplification, the fluorophore and quencher get separated from each other and fluorescence can be detected (Schaad and Frederick, 2002).

2.3.     Scorpion-PCR

This assay employs two primers, one of which serves as a probe and contains a stem-loop structure with a fluorescent reporter at 5' end and 3' quencher at 3' end. The sequence of loop of the Scorpions probe is complementary to an internal segment of the target sequence on the same strand. During the first amplification cycle, the Scorpions primer is extended and the sequence complementary to the loop sequence is generated on the same strand. The Scorpions probe contains a PCR blocker just 3' of the quencher to prevent read-through during the extension of the opposite strand. After subsequent denaturation and annealing, the loop of the Scorpions probe hybridizes to the target sequence by an intra-molecular interaction, and the reporter is separated from the quencher. The resulting fluorescent signal is proportional to the amount of amplified product in the sample. Comparison among Scorpion primers and alternative chemistries showed that TaqMan probes have high backgrounds and moderate signal strength, molecular beacons have low background fluorescence and low signal strength, Scorpion primers have low backgrounds and high signal strength (Thelwell et al., 2000).

Continued......