Filipiak, A.

Tomalak, M.

English

JKI Open Journal Systems (Julius Kühn-Institut)

  IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 133 Filipak A, Tomalak M, The use of PCR-HRM technique for detection of the quarantine nematode Bursaphelenchus xylophilus. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 133-135, Braunschweig, ISSN: 1866-590X (162) The use of PCR-HRM technique for detection of the quarantine nematode Bursaphelenchus xylophilus Anna FilipiakDepartment of Biological Pest Control, Institute of Plant Protection – National Research Institute, ul. Wladyslawa Wegorka 20, 60-318 Poznan, Poland. , Marek Tomalak,  Email: A.Filipiak@iorpib.poznan.pl INTRODUCTION High Resolution Melting Analysis (PCR-HRM) is a recently developed technique for fast, high-throughput post-PCR analysis of genetic mutations or variance in nucleic acid sequences. It enables researchers to detect rapidly and categorize genetic mutations, identify new genetic variants without sequencing, or determine the genetic variation in a population prior to sequencing (Pasay et al., 2008). We have examined this technique for detection and distinguishing of the quarantine pest, B. xylophilus from the morphologically and genetically most similar nematode B. mucronatus. MATERIALS AND METHODS Genomic DNA of B. xylophilus (isolate China) and B. mucronatus (isolate Wro-01) (about 100 nematodes) was isolated with QIAamp DNA Micro Kit (QIAGEN) according to the protocol provided by the manufacturer. For each species the isolation was conducted separately. The specific primers were designed from the ITS-1 region (forward: 5’- CGTGCAACGGTAAAGTCTGGGTTT-3’ and reverse 5’- AATCCTACGCTCGCCAGAACGAAT-3’) (Fig. 1). The PCR product was expected to be 112 bp in length. The PCR-HRM assays were performed with the use of Rotor–Gene Q Thermocycler (Qiagen). The obtained data was analysed according to the manufacturer’s instructions.  A              ....|....| ....|....| ....|....| ....|....| ....|....|                       160        170        180        190        200         BmWro-01     CGCACGGAAG CCGAGAGGTG ACCGTGCAAC GGTAAAGTCT GGGTTTBx           CGCATGGAAG CCGAGAGGCG ACCGTGCAAC GGTGAAGTCT GGGTTTCTAC  CTAT   B              ....|....| ....|....| ....|....| ....|....| ....|....|                       260        270        280        290        300         BmWro-01     GCTGCTTGCC GATTCGTTCT GGCGAGCGTA GGATTBx           GCTGCTTGCC GATTCGTTCT GGCGAGCGTA GGATTGAAAA GCCCGAGAGG  GAAAA GCCCGAGCGG     IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 134 Fig. 1. Diagrams indicating differences in sequence alignment of the forward primer (A) and reverse primer (B) between B. xylophilus and B. mucronatus.  RESULTS AND DISCUSSION The study conducted with the designed primers allowed us to distinguish and identify two morphologically similar nematode species, i.e. B. xylophilus and B. mucronatus. Melting curve analysis of the reaction products showed the presence of a single amplification product for each of the performed reactions (Fig. 2). The graph shows mean values from four replicates of each reaction.    Fig. 2. Melting curves of PCR-HRM products: B. xylophilus and B. mucronatus. The vertical axis shows the fluorescence value (dF/dT), and the horizontal axis the value of the melting temperature (Tm) (°C).   The normalized DNA melting curves obtained in the HRM analysis differed from each other in denaturation temperature, as evidenced by a substantial shift of these curves in relation to each other. This indicates the differences between examined species in the composition of nucleotides within the amplified region of the genome. The analogous differences are also evident in the normalized differentiating graph (Fig. 3).   Fig. 3. Normalized differentiating graph of PCR-HRM products for: B. xylophilus and B. mucronatus.        IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 135 Conducted study confirmed the high efficiency of the designed primers to distinguish the quarantine nematode B. xylophilus from the most closely related B. mucronatus. Since the PCR-HRM reaction allows for detection of single changes in nucleotides in tested of PCR products, it seems to be a very promising method for supporting identification decisions in the case of closely related species and isolates which could give ambiguous results in the real-time PCR reaction. Compared to the other molecular methods, PCR-HRM technique can be much simpler and less expensive way to identify the quarantine nematode B. xylophilus. REFERENCES Pasay C, Arlian L, Morgan M, Vyszenski-Moher D, Rose A, Holt D, Walton S, McCarthy J. (2008). High-resolution melt analysis for the detection of a mutation associated with permethrin resistance in a population of scabies mites. Medical and Veterinary Entomology 22, 82–88.  

The use of PCR-HRM technique for detection of the quarantine nematode Bursaphelenchus xylophilus

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The use of PCR-HRM technique for detection of the quarantine nematode Bursaphelenchus xylophilus

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