Optimisation of a species-specific primer set to quantify the soybean cyst nematode, Heterodera glycines, in soil using real-time PCR

Abstract

SummaryWe previously reported a real-time PCR primer set (SCN) that is specific to the soybean cyst nematode Heterodera glycines, a major nematode pest in soybean production in Japan. However, the primer set also amplified the related species H. trifolii and H. schachtii, whose presence was recently reported in Japan. The objective of this study was to optimise a primer set to be more specific for quantification of H. glycines. The newly optimised primer set (SCNnew) amplified H. trifolii and H. schachtii at amplification efficiencies less than 1% of H. glycines. Surveys for H. glycines in different green soybean fields in Japan demonstrated that most fields judged to contain low densities of H. glycines based on the SCN primer set were not actually infested with H. glycines. The SCNnew primer set quantifies H. glycines in soil more precisely.

Evaluation of Soil Suppressiveness of Various Japanese Soils against the Soybean Cyst Nematode Heterodera glycines and Its Relation with the Soil Chemical and Biological Properties

“…We performed quantitative PCR using a StepOne Real-Time PCR System (Applied Biosystems Japan Ltd., Tokyo, Japan) with a final volume of 10 µL containing 2.0 µL of 10 times diluted template DNA, 0.4 µL of 10 µM each primer specific to H. glycines [36] (SCNnew-f (5 -CTGCACATGTGAAAGGTGTA-3 ) and SCNnew-r (5 -GAGCGTGCATCCATTG-3 )) and 5.0 µL of Fast SYBR ® Green Master Mix (Applied Biosystems Japan Ltd., Tokyo, Japan) under the manufacturer's recommended conditions (95 • C for 10 s, then 45 cycles of 95 • C for 5 s and 60 • C for 20 s, followed by a melt curve analysis (from 60 • C to 95 • C). In this study, a calibration curve was newly prepared for the infested soil by inoculating different numbers (400, 1600, and 6400) of eggs into the 20 g of the infested soil and extracting DNA from the inoculated soils with the method described in Section 2.2.…”

Suppression of the soybean cyst nematode, Heterodera glycines, by short-term field cultivation and soil incorporation of mung bean

“…Purified DNA extracts were used as templates in real-time PCR after ten-fold dilution. Real-time PCR was performed using a Step One Real-Time PCR System (Life Technologies Japan) in a final volume of 10 μl containing 5 μl of a Fast SYBR Green Master Mix (Life Technologies Japan), 5 mM of each primer (SCNnew-f (ITS1, 5'-CTG CAC ATG TGA AAG CCT GTG TA-3') and SCNnew-r (ITS1, 5'-GAG CGT GCA TCC CAC ATT G-3')) (Shirai & Toyota, 2019) and 2 μl of template DNA under the manufacturer's recommended conditions (95°C for 10 s, (95°C for 5 s and 60°C for 20 s) × 40 cycles). Ct values (y) obtained with the primer set were converted to the densities (x: log10 eggs equivalent (20 g dried soil) −1 ) of SCN using the calibration curve (y = -2.82x + 34.5) (Shirai & Toyota, 2019).…”

テンサイシストセンチュウ汚染土壌からの迅速なdna検出方法

“…DNA has also been extracted from soil samples. Species-specific primers were used in combination with realtime PCR to quantify H. glycines in 30 g samples of dried soil pulverized with a ball mill (Shirai and Toyota, 2019).Methods of extracting DNA from soil samples and identifying eukaryotic organisms in these samples include environmental DNA sequencing, also called metabarcoding. Soil samples are immersed in saturated phosphate buffer (0.12 M Na 2 HPO 4 /NaH 2 PO 4 , pH 8.0) to release DNA bound to soil…”

“…DNA has also been extracted from soil samples. Species-specific primers were used in combination with realtime PCR to quantify H. glycines in 30 g samples of dried soil pulverized with a ball mill (Shirai and Toyota, 2019).…”