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).

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