Study of optimal method of Heterodera glycines sample preparation for molecular diagnostics

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Abstract

In this study, it is optimized by the methods of preparing samples for the release of genetic material (DNA) from soybean cyst-forming nematode Heterodera glycines , which is necessary for accurate identification. Various modifications of ready-made reagent kits from Russian and foreign manufacturers were tested: DNA-Extrane-2, FitoSorb, TsytoSorb, ORB-GMO-A by Sintol LLC (Russia), Proba-GS, Proba-NK by AgroDiagnostika LLC, and QIAamp DNA Mini Kit by Qiagen (Germany). By testing the PCR method «In real time», the sensitivity of Heterodera glycines total DNA release was tested using a test system consisting of forward and reverse primers and a modified probe: SCNrtF (5’-AAATTCCAGGCCGCTATCTC-3’), SCNrtR (5’CGTGGACTGAACTGAACTGGACAAAG-3’), SCNrtP (5’-FAM/-TGGGCTGGGTGCTTCTAGAACTTTT-/ BHQ-1/-3’).

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Table 1
Amplification conditions and the composition of the reaction mixture

 Stage

 Temperature, 0C

 Time

 Cycles

 Initial denaturation

 95

 2 min

 1

 Denaturation

 95

 10 secs

 50

 Anneal primers (with fluorescence detection in the FAM channel)

 62

 45 secs

 

 The composition of the reaction mixture

 Volume of component per sample, μl

 5Х qPCRmix-­HS Evrogen

 5

 Forward primer SCNrtF (10 ρmol/μl)

 1

 Reverse primer SCNrtR (10 ρmol/μl)

 1

 Probe SCNrtP (5 ρmol/μl)

 1

 Sterile dH2O

 16

 DNA sample

 1

Source: compiled by A.V. Ivanov.

Table 2
Determining the concentration of DNA isolated from Heterodera glycines

 № п/п

 Reagent kit

 Sample

 Concentration, μg/mL

 The ratio of the 260/280 nm

 1

 DNA-Extran‑2 (Sintol)

 H. glycines (1)

 6.5

 1.94

 2

 H. glycines (2)

 3.1

 1.88

 3

 H. glycines (3)

 3.7

 1.91

 4

 H. glycines (4)

 5.6

 1.87

 5

 H. glycines (5)

 3.5

 1.81

 6

 Sorb-­GMO-A (Sintol)

 H. glycines (6)

 2.5

 1.59

 7

 H. glycines (7)

 2.8

 1.48

 8

 H. glycines (8)

 2.4

 1.49

 9

 H. glycines (9)

 3.5

 1.38

 10

 H. glycines (10)

 1.6

 1.59

 11

 Proba-­GS (Agrodiagnostika)

 H. glycines (11)

 11.1

 6.26

 12

 H. glycines (12)

 12.5

 5.54

 13

 H. glycines (13)

 10.5

 5.87

 14

 H. glycines (14)

 11.7

 8.40

 15

 H. glycines (15)

 10.7

 7.74

 16

 PhytoSorb (Sintol)

 H. glycines (16)

 14.4

 1.50

 17

 H. glycines (17)

 18.7

 1.44

 18

 H. glycines (18)

 17.4

 1.37

 19

 H. glycines (19)

 20.4

 1.40

 20

 H. glycines (20)

 17.5

 1.46

 21

 QIAamp DNA Mini Kit (Qiagen)

 H. glycines (21)

 0.7

 0.97

 22

 H. glycines (22)

 1.1

 0.98

 23

 H. glycines (23)

 1.2

 0.99

 24

 H. glycines (24)

 0.8

 1.14

 25

 H. glycines (25)

 1.6

 1.35

 26

 Proba-­NK (Agrodiagnostika)

 H. glycines (26)

 6.9

 1.37

 27

 H. glycines (27)

 4.9

 1.18

 28

 H. glycines (28)

 9.7

 1.44

 29

 H. glycines (29)

 7.0

 1.78

 30

 H. glycines (30)

 5.1

 1.31

 31

 CytoSorb (Sintol)

 H. glycines (31)

 29.6

 1.47

 32

 H. glycines (32)

 29.4

 1.50

 33

 H. glycines (33)

 27.1

 1.45

 34

 H. glycines (34)

 27.9

 1.45

 35

 H. glycines (35)

 30.6

 1.47

Source: compiled by A.V. Ivanov.

Table 3
The values of the threshold cycles of PCR-RT for DNA samples isolated from H. glycines

 Sample

 Reagents and the threshold cycle Ct fluorescent signal FAM,  specific for H. glycines SCN-SCAR marker

 DNA-Extran‑2 (Sintol)

 Sorb-­GMO-A (Sintol)

 Proba-­GS (Agro-diagnostika)

 PhytoSorb (Sintol)

 QIAamp DNA Mini Kit (Qiagen)

 Proba-­NK(Agro-diagnostika)

 CytoSorb (Sintol)

 H. glycines (1*)

 21.28

 41.68

 25.21

 25.45

 28.92

 21.98

 26.39

 H. glycines (2*)

 25.05

 30.29

 26.09

 25.37

 29.61

 23.05

 23.24

 H. glycines (3*)

 24.16

 27.22

 22.84

 25.08

 26.06

 25.70

 23.35

 H.glycines (4*)

 21.24

 41.56

 23.44

 24.70

 27.14

 21.81

 27.36

 H.glycines (5*)

 24.21

 28.09

 26.92

 23.99

 26.99

 25.35

 25.39

 К-

 N/A

 N/A

 N/A

 N/A

 N/A

 N/A

 N/A

Av.v. ± St.er.

 23.19 ± 0.8

 33.79 ± 3.2

 24.90 ± 0.7

24.92 ± 0.3

27.74 ± 0.6

 23.58 ± 0.8

 25.15 ± 0.8

Note. К- — negative control sample; N/A — no fluorescence signal; Av. v. — average value; St. er. — standard error.
Source: compiled by A.V. Ivanov.

×

About the authors

Anton V. Ivanov

All-Russian Plant Quarantine Center

Author for correspondence.
Email: tonijons8@mail.ru
ORCID iD: 0009-0002-5361-6100
SPIN-code: 4537-6755

Junior researcher, Helminthology Laboratory, Testing Laboratory

32 Pogranichnaya st., Bykovo, 140150, Moscow region, Russian Federation

Galina N. Bondarenko

All-Russian Plant Quarantine Center

Email: reseachergm@mail.ru
ORCID iD: 0000-0002-1635-2508
SPIN-code: 2631-5209

Candidate of Biological Sciences, Senior researcher, Head of the Testing Laboratory Center

32 Pogranichnaya st., Bykovo, 140150, Moscow region, Russian Federation

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Copyright (c) 2025 Ivanov A.V., Bondarenko G.N.

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