A reliable and efficient BioPulverizer method in preparing and grinding nematodes for nucleic acid extraction and molecular identification

Due to their tiny size, soil nematodes and plant-parasitic nematodes are challenging to homogenize and collect using traditional mortar and pestle methods. To overcome this, we developed a reliable and efficient nematode preparation and grinding method using the BioPulverizer. The method involves creating a modified sample ‘sandwich’ with liquid nitrogen-frozen nematode samples placed between two layers of aluminum foil. This ‘sandwich’ facilitates complete homogenization through cryogenic grinding without any sample loss during transfer. The powerful nematode grinding yields high-quality and high-quantity samples, suitable for nucleic acid release and subsequent molecular identification and other downstream applications.

KEY WORDS:
Cryogenic grinding; homogenization; plant parasitic; soil nematodes

Authorship

Ye Jiang

Data curation

Formal analysis

Methodology

Investigation

Writing - original draft

State Key Laboratory of Black Soils Conservation and Utilization, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. Harbin 150081, Heilongjiang, China.Chinese Academy of SciencesChinaHeilongjiang, ChinaState Key Laboratory of Black Soils Conservation and Utilization, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. Harbin 150081, Heilongjiang, China.

University of Chinese Academy of Sciences. Beijing 100049, China.University of Chinese Academy of SciencesChinaBeijing, ChinaUniversity of Chinese Academy of Sciences. Beijing 100049, China.

http://orcid.org/0009-0009-7724-9166

Ruifeng Qin

Investigation

University of Chinese Academy of Sciences. Beijing 100049, China.University of Chinese Academy of SciencesChinaBeijing, ChinaUniversity of Chinese Academy of Sciences. Beijing 100049, China.

http://orcid.org/0009-0006-8537-7043

Chunjie Li

Resources

Data curation

http://orcid.org/0000-0002-2847-1504

Minghui Huang

Investigation

http://orcid.org/0009-0008-0884-6226

Dan Jiang

Investigation

University of Chinese Academy of Sciences. Beijing 100049, China.University of Chinese Academy of SciencesChinaBeijing, ChinaUniversity of Chinese Academy of Sciences. Beijing 100049, China.

http://orcid.org/0000-0002-0840-1760

Doudou Chang

Investigation

University of Chinese Academy of Sciences. Beijing 100049, China.University of Chinese Academy of SciencesChinaBeijing, ChinaUniversity of Chinese Academy of Sciences. Beijing 100049, China.

http://orcid.org/0009-0008-3937-8365

Yifan Xie

Investigation

University of Chinese Academy of Sciences. Beijing 100049, China.University of Chinese Academy of SciencesChinaBeijing, ChinaUniversity of Chinese Academy of Sciences. Beijing 100049, China.

http://orcid.org/0009-0003-1443-2437

Yanan Zhao

Investigation

http://orcid.org/0009-0001-0768-8093

Congli Wang

Conceptualization

Resources

Data curation

Supervision

Validation

Metho dology

Project administration

Writing - review & editing

http://orcid.org/0000-0002-3318-4171

Corresponding author: Congli Wang (wangcongli@iga.ac.cn)

Editorial responsibility

Walter A.P. Boeger

Competing Interests

The authors have declared that no competing interests exist.

SCIMAGO INSTITUTIONS RANKINGS

University of Chinese Academy of Sciences. Beijing 100049, China.University of Chinese Academy of SciencesChinaBeijing, ChinaUniversity of Chinese Academy of Sciences. Beijing 100049, China.

Figures

Figures (2)

Thumbnail

Figure 1
Equipment used for nematode preparation and grinding. The names of all the equipment are listed above or under each respective one.

Thumbnail

Figures 2-6
PCR amplification product with primers: (2) PCR products from the seminested primer pairs (NemF and 18Sr2b; NF1 and 18Sr2b) obtained from soil nematode samples with BioPulverizer grinding; (3) PCR amplification with the first cycle of the primer pair (NemF and 18Sr2b) from soil samples without BioPulverizer grinding; (4-5) Two amplification bands, 181 bp with the species-specific primer pair GlyF1/rDNA2 (4) and 477 bp with SCNF1/SCNR1 (5), were amplified for Heterodera glycines; (6) PCR products with the universal primer pair 194F/195R and the species-specific primer pair (Meloidogyne incognita) from potato tuber samples. All molecular markers (M) are 100 bp DNA ladders. The concentrations of agarose gel are 1.8% in Figs 2-3 and 1% in Figs 4-6.

Figure 1 Equipment used for nematode preparation and grinding. The names of all the equipment are listed above or under each respective one.

Figures 2-6 PCR amplification product with primers: (2) PCR products from the seminested primer pairs (NemF and 18Sr2b; NF1 and 18Sr2b) obtained from soil nematode samples with BioPulverizer grinding; (3) PCR amplification with the first cycle of the primer pair (NemF and 18Sr2b) from soil samples without BioPulverizer grinding; (4-5) Two amplification bands, 181 bp with the species-specific primer pair GlyF1/rDNA2 (4) and 477 bp with SCNF1/SCNR1 (5), were amplified for Heterodera glycines; (6) PCR products with the universal primer pair 194F/195R and the species-specific primer pair (Meloidogyne incognita) from potato tuber samples. All molecular markers (M) are 100 bp DNA ladders. The concentrations of agarose gel are 1.8% in Figs 2-3 and 1% in Figs 4-6.

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A reliable and efficient BioPulverizer method in preparing and grinding nematodes for nucleic acid extraction and molecular identification

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[1] Corresponding author: Congli Wang (wangcongli@iga.ac.cn)