Using microchip-based qPCR for the Rapid Detection of Potato Pathogens

A quick, robust, cost-effective, and ready-to-use microchip diagnostic tool was developed for the accurate and sensitive detection of potato pathogens. This tool may prove advantageous to field-men, growers, pathogen testing labs, extension personnel, and seed potato storage facilities to make an accurate disease assessment.

Using qPCR for the Rapid Detection of Potato Pathogens

In order to minimize disease-induced potato losses during production and storage, the advanced detection of potato diseases is needed. In addition to the detection of latent or masked infections, sensitive and accurate detection of potato pathogens, discrimination among visually complicated co-infections is crucial [1]. Furthermore, accurate diagnostics can supply proper guidance for the management of the disease, avoiding irrelevant and unnecessary pesticide applications.

Yet, in the instance of climatic changes influencing plant resistance to pathogens, plus the invasion of the pathogens into new areas and global trade inducing pathogen behavior with plants, fast and early disease diagnostics become extremely important [2].

Considering these limitations, this article presents a convenient and easy tool that provides sensitive, cost-effective, quick, and specific multiplex diagnostics of a large range of potato pathogens.

The diagnostic tool is based on the polymerase chain reaction (PCR). Among the advanced molecular-based detection methods, PCR amplifies the specific genes of the pathogens in their DNA/RNA samples. It provides more accurate, sensitive, and quicker identification than the protein detecting methods like ELISA and lateral flow devices that are unable to detect viroids, which are potent pathogens [3].

The simultaneous detection and identification of many potato pathogens can be performed by utilizing a 30-well microchip via an AriaDNA PCR analyzer

In the development of this diagnostic system, the master mixes of the test kits for the relevant panel of potato pathogens were stabilized and freeze-dried into the microchips, in order to ease the test process.

The primer-probe oligonucleotide sets of the whole panel of potato pathogens were analyzed for their specificity. Diagnostic efficiency of test kits with lyophilized and immobilized PCR master-mixes varied insignificantly from that of freshly prepared test kits. The sensitivity of the developed test systems differed from one to five copies of the pathogen genome present in the DNA/RNA samples added to the microreactor.

The technology provides high reproducibility within 0.2 – 1.0 % (standard deviation, n=5). The automated data interpretation and the simplicity of the analysis provides high-throughput monitoring and surveillance of the potato pathogens.

Table 1.

  PVY PVY o PVY ntn PVX PVA PVM PVS PMTV PSTVd
Sample 1
Sample 2
Sample 3
C+
C-
IC


Figure 2. Typical results of qPCR from potato tuber samples performed in a ready-to-run microchip reporting potato pathogens Phytophtora infestans (P. inf) and Ralstonia solanacearum (Rs). The figure also displays positive control samples C+ (purple curves), negative control samples C‒ (orange curves) and internal control sample (IC).

Figure 3. Layout of the DNA Pathogens Microchip Kit (left) and RNA Pathogens Microchip Kit (right).

Benefits of this technology

  • High efficiency: In one microchip, a full panel of pathogens may be tested in one sample or 28 samples can be tested against 1 target.
  • Simple: No need to prepare master mixes since everything is already dried in the microchip. It only needs to add DNA/RNA samples into the microchip.
  • Customizable: The panel of test pathogens and their layout in the microchip can be customized:
    • 1 pathogen test kit: 28 samples
    • A whole panel of pathogens test kits: Lesser number of samples
  • Cost-effective: Each data point is in 1.2 µl reaction, and so, the test cost would be lower than the traditional real-time PCR.
  • Quick results: Faster results (between 20 and 30 minutes) than any other qPCR
  • Empty or ready-to-run microchips:
    • Ready-to-run microchips: Have freeze-dried reagents
    • Empty microchips: To run 1.2 µl reaction with liquid reagents
  • Reporting: Qualitative (+/-) and Quantitative (Ct values)
  • Confidence in test results: Being 1.2 µl reaction, it is highly sensitive, specific, and robust and able to report one to five copies of the test pathogen.
  • Shipping conditions: Cold-chain free transportation
  • Shelf-life: The shelf life of ready-to-run microchips is up to a year

References and Further Reading

  1. De Haan, et al., Potato Res. 2005, 48:1–14
  2. Elad, Y., et al., Crop. Improv. 2014, 28:99–139
  3. Martinelli, F., et al., Agron. Sustain. Dev. 2015, 35:1–25
  4. Nikitin, M., et al., Biosensors 2018, 8:129–147

About Lumex Instruments

For almost 30 years, Lumex Instruments has been developing and manufacturing laboratory and industrial analytical equipment, instruments, and procedures.

Our products are used for environmental control, water and wastewater treatment, agriculture, food and beverage quality and safety control, energy industry, and pharmaceutical and biochemical applications. Lumex Instruments offers more than 120 applications for the measurement of organic and inorganic chemicals in liquid, solid and gas.

We offer both traditional and new ingenious solutions, unique technologies and classical equipment, well-established reliable applications and innovative methods. From the beginning of the company, we have developed, manufactured and sold more than 16,500 instruments.

Our products include:

  • capillary electrophoresis systems
  • PCR analyzers
  • atomic absorption spectrometers (including mercury analyzers and monitors)
  • FTIR and NIR Spectrometers
  • fluorimeters and more

We combine high-quality standards with the flexible customized approach because we care about the efficiency of our customer operations. All our innovations are targeted to make the analyses cost-efficient, easy to run, and the instruments easy to maintain and service. We proudly deliver our products and services all around the world and we enjoy working with our valued customers and partners in many industries and countries.

With customers in more than 90 countries worldwide, Lumex Instruments has several offices and multiple distributors around the world.

A significant number of our applications are developed in accordance with international standards, such as ASTM, ISO, EN, and EPA.

Facts and figures:

  • More than 30 models of analytical instruments developed starting from 1991
  • Over 20 patents
  • 20% market share of the portable mercury analyzers
  • Over 120 applications
  • Over 350 employees, and 25% of them are involved in the development of new applications and instruments

The customers who selected Lumex Instruments include BP, Enel, Royal Dutch Shell, PETRONAS, PetroChina, Chevron, UNIDO, SGS, Carlsberg Group, Unilever, PepsiCo, Nestle, Princeton University, and others.


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Last updated: Jul 24, 2020 at 5:26 AM

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