Open Access

Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease

L.K. Carta
L.K. Carta
 and 
S. Li
S. Li
   | Mar 17, 2020
Journal of Nematology's Cover Image
About this article
Previous Article
Next Article
Cite
Published Online: Mar 17, 2020
Page range: 1 - 15
Received: Sep 30, 2019
DOI: https://doi.org/10.21307/jofnem-2020-016
Keywords
© 2020 L.K. Carta et al., published by Sciendo.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 7:

Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with both DreamTaq™ and Pfu in manufacturer’s PCR buffers. M: DNA markers; 1: 104K29; 2: 104K30; 3: 104K31; NC: negative control, respectively. A: DreamTaq™ PCR buffer; B: Pfu PCR buffer.
Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with both DreamTaq™ and Pfu in manufacturer’s PCR buffers. M: DNA markers; 1: 104K29; 2: 104K30; 3: 104K31; NC: negative control, respectively. A: DreamTaq™ PCR buffer; B: Pfu PCR buffer.

Figure 1:

Long range ribosomal PCR Amplifications of the 3.5 kb target from Fall specimens with DreamTaq™. M: DNA markers; 1: 104H78; 2: 104H81; 3: 104H82; 4: 104H83; 5: 104H84; 6: 104H85; 7: 104H86; 8: 104H87; 9: 104H88; 10: 104H89; 11: 104H90; NC: negative control. 1-7: Female; 8-11: Male.
Long range ribosomal PCR Amplifications of the 3.5 kb target from Fall specimens with DreamTaq™. M: DNA markers; 1: 104H78; 2: 104H81; 3: 104H82; 4: 104H83; 5: 104H84; 6: 104H85; 7: 104H86; 8: 104H87; 9: 104H88; 10: 104H89; 11: 104H90; NC: negative control. 1-7: Female; 8-11: Male.

Figure 2:

Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with TaKaRa Ex Taq® system. M: DNA markers; 1: 104J54; 2: 104J55; 3: 104J58; 4: 104J59; NC: negative control, respectively. A: DreamTaq™; B: 18 S locus (1.7 kb) by DreamTaq™, C: ITS and 28 S loci (1.9 kb) by DreamTaq™; D: TaKaRa Ex Taq® system.
Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with TaKaRa Ex Taq® system. M: DNA markers; 1: 104J54; 2: 104J55; 3: 104J58; 4: 104J59; NC: negative control, respectively. A: DreamTaq™; B: 18 S locus (1.7 kb) by DreamTaq™, C: ITS and 28 S loci (1.9 kb) by DreamTaq™; D: TaKaRa Ex Taq® system.

Figure 3:

Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K17; 2: 104K18; 3: 104K19; 4: 104K20; NC: negative control, respectively. A: DreamTaq™; B: PicoMaxx™ High Fidelity PCR System.
Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K17; 2: 104K18; 3: 104K19; 4: 104K20; NC: negative control, respectively. A: DreamTaq™; B: PicoMaxx™ High Fidelity PCR System.

Figure 4:

Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: DreamTaq™; B: PicoMaxx™ High Fidelity PCR System.
Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: DreamTaq™; B: PicoMaxx™ High Fidelity PCR System.

Figure 9:

PCR performance of TaKaRa Ex Taq® system and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. A: 1, 2, 3 and 4: TaKaRa Ex Taq® system; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System; B: 1, 2, 3 and 4: TaKaRa Ex Taq® system and DreamTaq™; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System. NC: negative control, respectively.
PCR performance of TaKaRa Ex Taq® system and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. A: 1, 2, 3 and 4: TaKaRa Ex Taq® system; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System; B: 1, 2, 3 and 4: TaKaRa Ex Taq® system and DreamTaq™; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System. NC: negative control, respectively.

Figure 5:

Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with DreamTaq™ and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: 18 S locus (1.7 kb) by DreamTaq™, B: ITS and 28 S loci (1.9 kb) by DreamTaq™; C: DreamTaq™ and PicoMaxx™ High Fidelity PCR System combined.
Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with DreamTaq™ and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: 18 S locus (1.7 kb) by DreamTaq™, B: ITS and 28 S loci (1.9 kb) by DreamTaq™; C: DreamTaq™ and PicoMaxx™ High Fidelity PCR System combined.

Figure 6:

Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with DreamTaq™ or/and Pfu in PicoMaxx™ buffer. M: DNA markers; 1, 2, 3 and 4: DreamTaq™; 5, 6, 7 and 8: Pfu; 9, 10, 11and 12: DreamTaq™ and Pfu combined; 1, 5 and 9: 104K29; 2, 6 and 10: 104K30; 3, 7 and 11: 104K31; 4, 8 and 12: negative control (NC), respectively.
Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with DreamTaq™ or/and Pfu in PicoMaxx™ buffer. M: DNA markers; 1, 2, 3 and 4: DreamTaq™; 5, 6, 7 and 8: Pfu; 9, 10, 11and 12: DreamTaq™ and Pfu combined; 1, 5 and 9: 104K29; 2, 6 and 10: 104K30; 3, 7 and 11: 104K31; 4, 8 and 12: negative control (NC), respectively.

Figure 8:

PCR performance of Pfu and Pwo in PicoMaxx™ buffer. M: DNA markers; 1 and 4: 104K37; 2 and 5: 104K38; 3 and 6: 104K39. A: 1, 2 and 3: DreamTaq™; 4, 5 and 6: Pwo (0.125 μl per reaction). B: 1, 2 and 3: DreamTaq™ and Pfu; 4, 5 and 6: DreamTaq™ and Pwo (0.125 μl per reaction). NC: negative control, respectively. Note: final concentration of Pfu in each reaction was aligned with Pwo and DreamTaq™ in 0.625 units.
PCR performance of Pfu and Pwo in PicoMaxx™ buffer. M: DNA markers; 1 and 4: 104K37; 2 and 5: 104K38; 3 and 6: 104K39. A: 1, 2 and 3: DreamTaq™; 4, 5 and 6: Pwo (0.125 μl per reaction). B: 1, 2 and 3: DreamTaq™ and Pfu; 4, 5 and 6: DreamTaq™ and Pwo (0.125 μl per reaction). NC: negative control, respectively. Note: final concentration of Pfu in each reaction was aligned with Pwo and DreamTaq™ in 0.625 units.

Figure 10:

PCR performance of Herculase® II Fusion DNA polymerase and Phusion™ High-Fidelity DNA Polymerase. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. 1, 2, 3 and 4: Herculase® II Fusion DNA polymerase; 5, 6, 7 and 8: Phusion™ High Fidelity PCR System; NC: negative control, respectively.
PCR performance of Herculase® II Fusion DNA polymerase and Phusion™ High-Fidelity DNA Polymerase. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. 1, 2, 3 and 4: Herculase® II Fusion DNA polymerase; 5, 6, 7 and 8: Phusion™ High Fidelity PCR System; NC: negative control, respectively.

Figure 11:

PCR performance of Taq2000™, Platinum™ Taq and DreamTaq™. M: DNA markers; 1, 4 and 7: 104N95; 2, 5 and 8: 104N96; 3, 6 and 9: 104N97. 1, 2, 3 and NC by Taq2000™; 4, 5, 6 and NC by Platinum™ Taq; 7, 8, 9 and NC by DreamTaq™, NC: negative control, respectively. A: 3.5 kb target; B: 1.9 kb ITS and 28 S target. Note: final concentration of either Taq2000™ or DreamTaq™ in each reaction was aligned with Platinum™ Taq in 1.25 units.
PCR performance of Taq2000™, Platinum™ Taq and DreamTaq™. M: DNA markers; 1, 4 and 7: 104N95; 2, 5 and 8: 104N96; 3, 6 and 9: 104N97. 1, 2, 3 and NC by Taq2000™; 4, 5, 6 and NC by Platinum™ Taq; 7, 8, 9 and NC by DreamTaq™, NC: negative control, respectively. A: 3.5 kb target; B: 1.9 kb ITS and 28 S target. Note: final concentration of either Taq2000™ or DreamTaq™ in each reaction was aligned with Platinum™ Taq in 1.25 units.

Summary of PCR performance of blended DNA polymerases (systems) tested in this study.

TaKaRa Ex Taq™ combined with DreamTaq™ in TaKaRa Ex buffer PicoMaxx™ System combined with DreamTaq™ in PicoMaxx™ buffer DreamTaq™ in PicoMaxx™ buffer pfu in PicoMaxx™ buffer Pfu combined with DreamTaq™ in PicoMaxx™ buffer pfu combined with DreamTaq™ in DreamTaq™ buffer pfu combined with DreamTaq™ in pfu buffer Pwo in PicoMaxx™ buffer Pwo combined with DreamTaq™ in Pwo buffer Pwo combined with DreamTaq™ in PicoMaxx buffer
Spring specimens na na na na na na na na na na
Summer specimens 3.5 kb: X 3.5 kb: √√√/X 3.5 kb: √/X or X 3.5 kb: X 3.5 kb: √√√√ or √√ 3.5 kb: X 3.5 kb: X 3.5 kb: X 3.5 kb: X (DNS) 3.5 kb: √/X
Fall specimens na na na na na na na na na na

Summary of PCR performance of Individual DNA polymerases (systems) tested in this study.

Platinum™ Taq Taq2000™ DreamTaq™ TaKaRa Ex Taq™ PicoMaxx™ System pfu Pwo Herculase® II Phusion™
Spring specimens 3.5 kb: X; 1.9 kb: √/X 3.5 kb: X; 1.9 kb: √√√√/X 3.5 kb: X; 1.9 kb: √√√√ na na na na na na
Summer specimens na 3.5 kb: X (DNS); 1.9 kb: √√√ (DNS) 3.5 kb: X or X/√ (DNS); 1.7Kb: √√√√/X; 1.9 kb: √√√√/X 3.5 kb: √√/X or X 3.5 kb: √√/X or X 3.5 kb: X (DNS) 3.5 kb: X (DNS) 3.5 kb: X 3.5 kb: X
Fall specimens na na 3.5 kb: √√√/X; 1.9 kb: NA na na na na na na

Litylenchus crenatae specimens from American beech trees (Fagus grandifolia) with BLD tested in this study.

Specimens Locality Part Session
104H78, 104H81, 104H82, 104H83, 104H84, 104H85, 104H86, 104H87, 104H88, 104H89 and 104H90 Lake County, Ohio Leaf Fall (November, 2017)
104J54, 104J55, 104J56 and 104J57 Cuyahoga County, Ohio Leaf Summer (May, 2018)
104K17, 104K18, 104K19 and 104K20 The Holden Arboretum, Kirtland, Ohio Leaf Summer (August, 2018)
104K25, 104K26, 104K27, 104K28, 104K29, 104K30 and 104K31 Potter County, Pennsylvania Leaf Summer (August, 2018)
104K37, 104K38 and 104K39 Crawford County, Pennsylvania Leaf Summer (August, 2018)
104N95, 104N96 and 104N97 The Holden Arboretum, Kirtland, Ohio Bud Spring (March, 2019)

PCR cycling conditions.

No. Step pfu or combined with DreamTaq™ Herculase® II Phusion™ Pwo or combined with DreamTaq™
1. Initial denaturation 95°C for 2 min Step 1: 1 cycle 95°C for 2 min Step 1: 1 cycle 95°C for 2 min Step 1: 1 cycle 95°C for 2 min Step 1: 1 cycle
2. Denaturation 95°C for 30 sec Step 2, 3 and 4: 36 cycles 95°C for 20 sec Step 2, 3 and 4: 36 cycles 95°C for 20 sec Step 2, 3 and 4: 36 cycles 95°C for 30 sec Step 2, 3 and 4: 36 cycles
3. Annealling 55°C for 45 sec 55°C for 20 sec 55°C for 20 sec 57°C for 45 sec
4. Extension 72°C for 5 min 72°C for 2 min 15 sec 72°C for 2 min 15 sec 72°C for 5 min
5. Final extension 72°C for 7 min Step 5: 1 cycle 72°C for 7 min Step 5: 1 cycle 72°C for 7 min Step 5: 1 cycle 72°C for 7 min Step 5: 1 cycle

PCR components and setup.

Platinum™ Taq (10 units/μl) Taq2000™ (5 units/μl) DreamTaq™ (5 units/μl) TaKaRa Ex Taq™ (5 units/μl) or combined with DreamTaq™ (5 units/μl) PicoMaxx™ System (5 units/μl) or combined with DreamTaq™ (5 units/μl) pfu DNA polymerase (2.5 units/μl) or combined with DreamTaq™ (5 units/μl) Herculase® II Fusion DNA polymerase Phusion™ High-Fidelity DNA Polymerase (2 units/μl) Pwo DNA polymerase (5 units/μl) or combined with DreamTaq™ (5 units/μl)
Water (μl) Mixture A: 7.6
Water (μl) 15.375 16 16.375/16.25 15.875 or 15.75 17.3 or 17.175 17.05/17.55 or 16.925/17.425 16 14.5 Mixture B: 9.875 (or 9.75)
10 or 5x proprietary buffer (μl) 2.5 2.5 2.5 2.5 2.5 2.5 5 5 Mixture A: 2.5
50 mM MgCl2 (μl) 1 0.25
100 mM dNTP (25 mM each) (μl) 0.2 0.2 0.25 Mixture B: 0.4
10 mM dNTP (2.5 mM each) (μl) 2 0.5
8 mM dNTP (2 mM each) (μl) 2.5 2.5 2.5
10 μm Forward primer (μl) 0.75 0.75 0.75 1.25 1.25 1.25 0.625 1.25 Mixture B: 1.25
10 μm Reverse primer (μl) 0.75 0.75 0.75 1.25 1.25 1.25 0.625 1.25 Mixture B: 1.25
DMSO 0.25
DNA template (μl) 2 2 2 2 2 2 2 2 Mixture B: 2
Proprietary DNA polymerase(s) (μl) 0.125 0.25 0.125/0.25 0.125 or plus DreamTaq™: 0.125 0.5 or plus DreamTaq™: 0.125 0.75/0.25 or plus DreamTaq™: 0.125 0.5 0.25 Mixture A: 0.125 or plus DreamTaq™: 0.125
Total reaction volume (μl) 25 25 25 25 25 25 25 25 12.5 each mixture
eISSN:
2640-396X
Language:
English
Publication timeframe:
Volume Open
Journal Subjects:
Life Sciences, other
Journal RSS Feed