Development of a high throughput PPV detection method based on unique nucleic acid isolation system

By: Won-Sik Kim1 and Yousef Haj-Ahmad1,2, (1)Norgen Biotek Corp, 3430 Schmon Pkwy, Thorold, ON, L2V 4Y6, (2)Brock University, 500 Glenridge Ave., St. Catharines, ON, L2S 3A1

Product No. E4913

Abstract

Plum pox virus (PPV) is a serious plant disease affecting tender fruit including peaches, plums, apricots and nectarines, causing significant yield losses. Evidence suggests that the Dideron strain of PPV was first discovered in Ontario in 2000. Despite the rigid PPV eradication program that has been in place in Canada over the last decade, PPV still remains a threat to tender fruit growers in the Niagara region. PPV is transmissible from infected trees by aphids and it can take several years before virus titers are high enough or present in enough of the tree to be detectable by ELISA. This test is currently used by the Canadian Food Inspection Agency (CFIA) in the eradication program because of the assays specificity, low cost, high throughput and relatively simple methodology. Nevertheless, cross-reactivity, strain variability, as well as uneven and low virus titers in plant tissue have been associated with detection inconsistencies. Nucleic acid (NA) based detection has been proven to be more sensitive than immunological diagnostic methods; however, NAbased detection is more expensive and not as simple to implement. At Norgen Biotek Corp we have developed a NA-based PPV detection kit that is rapid, sensitive, inexpensive and simple to adopt high throughput robotic system. To improve the current PPV detection system, we have focused on the optimization of RNA purification, as sample preparation is the most critical step in any reliable diagnostic kit. Here we present In-house evaluation data on this NA-based PPV detection kit. We also anticipate to evaluate the PPV detection kit further with a large volume of field samples through a collaboration with a third party where regulatory pathogens (e.g. PPV) can be handled and analyized.

Introduction

Plum pox virus (PPV) is a member of the genus Potyvirus, and the virus is considered the most serious pathogen affecting stone fruits including apricots, cherry, nectarine, peaches, and plums. The symptoms of plum pox disease or Sharka include fruit abnormality, loss of taste, and fruits that drop prematurely. These symptoms may cause losses as high as 80–100% of a crop and consequently it is a disease of economic significance in every country where stone fruits are grown. To achieve successful eradicationor effective management of the associated disease, detection of the virus in infected plants is required.

As sample preparation is the most critical step in any reliable diagnostic analysis, Norgen’s proprietary resin showed outstanding affinity to purify all sizes of RNA from large mRNA and microRNA (miRNA) including small interfering RNA (siRNA) in comparison with a current market leader. This distinctive advantage has become a powerful tool contributing to a sensitive detection of virus and viroids. Single and 96 well plate RNA purification format has been developed with a compatibility with a robotic automation system for a high throughput analysis.

In order to provide a reliable, fast and cost effective PPV detection method, PPV RT-PCR Detection Kit was developed and evaluated in house with PPV transcripts expressed from the universal region of PPV RNA.

Results

Isolation of High Quality RNA, even from Difficult Samples

Figure 1. Isolation of High Quality RNA, even from Difficult Samples. Total RNA was isolated from 50 mg samples of apple (Lanes 1), peach (Lanes 2), grape (Lanes 3), pine needle (Lanes 4), strawberry (Lanes 5) and pear (Lanes 6) using Norgen’s kit and a competitors kit. Five µL of total RNA from the 50 µL of elution was loaded on 1X MOPS 1.0 % Formaldehyde-Agarose RNA gel for analysis. Norgen’s kit allowed for the isolation of high quality RNA from all the samples, including the difficult samples, while the competitor failed to isolate RNA from grape, pine needles and strawberry. Furthermore, only Norgen’s kit was able to isolate the small RNA species (white box).

Single column system   96 well plate & Robotic automation system


Figure 2. Norgen’s PPV RT-PCR Detection Kit is a ready-to-use system for the isolation and detection of PPV from plant samples. First, the kit contains components for the rapid isolation of total RNA, including viroid RNA, from the samples using spin-column chromatography based on Norgen’s proprietary resin. Second, the kit contains PPV Master Mix and controls to allow for PCR Amplification. The amplified PCR products are then detected using agarose gel electrophoresis. Alternatively, detection can be performed based on real-time PCR using melt curves.
OR



Figure 4. Interpretation of the Plum Pox RT-PCR Detection Kit. A representative 1X TAE 1.7% agarose gel showing the amplification of Plum Pox Virus at different concentrations (PPV Target). The size of the PPV target amplicon corresponds to 295 bp as represented by the provided RNA Marker (M). The size of the PPV Isolation Control (Iso C) corresponds to 498bp as represented by the provided RNA Marker (M). The PPV 2X PCR Master Mix contains a PPV PCR Control (PCRC). The PPV PCRC controls for PCR inhibition. The size of the PPV PCRC corresponds to 150bp as represented by the provided RNA Marker (M). The amplification from each lane is interpreted as shown below. Lane 1 to 4: Plum Pox Virus detected: Lanes showed the detection of all three PCR amplicons. Lane 5: No IsoC and Template– Only PPV PCRC was detected. Lane 6: PPV not detected: Detection of PPV IsoC and PPV PCRC, suggesting that the RNA isolation was successful but no PPV RNA was present in the sample. Lane 7: Positive: All three PCR amplicons were detected.

 

Norgen’s PPV RT-PCR detection kit is a ready-to-use system for the isolation and detection of PPV from PPV host samples

Figure 3. Norgen’s PPV RT-PCR detection kit is a ready-to-use system for the isolation and detection of PPV from PPV host samples. The PPV Master Mix contains reagents and enzymes for the specific amplification of a 295 bp region of the viral genome. In addition, PPV RT-PCR Detection Kit contains a second heterologous amplification system to identify possible PCR inhibition and/or inadequate isolations thereby eliminating false negatives.

Summary and Discussion

  • Proprietary resin isolates RNA from a wide range of samples than market leader.
  • PPV kit (or Plant/fungi total RNA purification kit) isolates a diversity of RNA species; All sizes of RNA are isolated from large mRNA down to microRNA without extra phenol or chloroform extraction.
  • Norgen’s PPV RT-PCR detection kit is easy to use, a simple and reproducible method for the isolation and detection of PPV from infected plant tissues.
  • Internal and isolation control in the PPV master mix give a accreditation to avoid any false positive or negative.
  • For high throughput analysis PPV RT-PCR detection kit is compatable with 96 well column plate and robotic automation system (Qiagen BioRobot 8000).
  • Industrial funding is available to evaluate Norgen’s PPV RTPCR PCR system