scholarly journals Application of a simple and high-throughput DNA extraction method to real-time PCR quantification of target plant-parasitic nematodes in nematode communities

2018 ◽  
Vol 48 (1) ◽  
pp. 1-10
Author(s):  
Masanori Kawanobe ◽  
Koki Toyota
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
High Throughput ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Parasitic Nematodes ◽  
Plant Parasitic Nematodes ◽  
Dna Extraction Method ◽  
Target Plant ◽  
Plant Parasitic

A novel nematode diagnostic method using the direct quantification of major plant-parasitic nematodes in soil by real-time PCR

Nematology ◽  
2012 ◽  
Vol 14 (3) ◽  
pp. 265-276 ◽  
Author(s):  
Yu Yu Min ◽  
Koki Toyota ◽  
Erika Sato
Keyword(s):  
Real Time ◽  
Sweet Potato ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Cyst Nematode ◽  
Parasitic Nematodes ◽  
Plant Parasitic Nematodes ◽  
Pcr Method ◽  
Plant Parasitic ◽  
Direct Quantification

We have developed a direct quantification method using real-time PCR for various plant-parasitic nematodes. Firstly, specific primers were designed for the root-knot nematode Meloidogyne incognita, the root-lesion nematode Pratylenchus penetrans, the potato cyst nematode Globodera rostochiensis and the soybean cyst nematode Heterodera glycines. A DNA extraction method was then developed beginning with 20 g of soil, a relatively large amount of soil but a necessary amount in the consideration of heterogeneous distribution of nematodes in soil. To estimate the density of the target nematode in soil, calibration curves for each plant-parasitic nematode were obtained by inoculating different numbers of the target nematode and then extracting DNA from the soils. The detection limit was 4-5 nematodes (20 g soil)−1. This method was applied to nematode diagnostics. Soil sampling was done when transplanting of radish and sweet potato in fields was taking place, and the density of plant-parasitic nematodes was measured using both the Baermann funnel extraction method and real-time PCR methods. In some soils, P. penetrans and M. incognita were not found with the Baermann method but were detected with the real-time PCR method. At harvest, damage to crops was evaluated and its relationship with initial densities was investigated. The real-time PCR method more precisely predicted damage to radish and sweet potato by nematodes and was considered to be a powerful tool in the diagnosis of nematode diseases.

Influence of Primer Sequences and DNA Extraction Method on Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in Ground Beef by Real-Time PCR Targeting the eae, stx, and Serogroup-Specific Genes†

2012 ◽  
Vol 75 (11) ◽  
pp. 1939-1950 ◽  
Author(s):  
JAMIE L. WASILENKO ◽  
PINA M. FRATAMICO ◽  
NEELAM NARANG ◽  
GLENN E. TILLMAN ◽  
SCOTT LADELY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Dna Extraction ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Ground Beef ◽  
Dna Extraction Method ◽  
Big Six ◽  
Pcr Targeting

Non-O157 Shiga toxin–producing Escherichia coli (STEC) infections, particularly those caused by the “big six” or “top six” non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx1, stx2, and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx1d, stx2e, and stx2g, are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx1d, stx2e, and stx2g, and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

Rapid detection of porcine DNA in processed food samples using a streamlined DNA extraction method combined with the SYBR Green real-time PCR assay

2020 ◽  
Vol 309 ◽  
pp. 125654 ◽  
Author(s):  
Lee Lee Tan ◽  
Siti Aminah Ahmed ◽  
Siew Kit Ng ◽  
Marimuthu Citartan ◽  
Carsten A. Raabe ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Extraction Method ◽  
Food Samples ◽  
Sybr Green ◽  
Processed Food ◽  
Pcr Assay ◽  
Dna Extraction Method

scholarly journals A new, harmless, high-throughput endosperm-based DNA extraction method for wheat

2019 ◽  
Vol 49 (9) ◽  
Author(s):  
Zhihui Ma ◽  
Yuquan Wang ◽  
Wenhui Wei ◽  
Zhengang Ru
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
Seedling Emergence ◽  
Control Group ◽  
Suitable Alternative ◽  
Dna Extraction Method ◽  
High Throughput Method ◽  
Young Leaves ◽  
Non Destructive

ABSTRACT: In this study, a non-destructive, high-throughput, endosperm-based DNA extraction method was developed. To verify the non-destructive nature of this method, a germination test was performed on 288 seeds after sampling their endosperm, which gave a seedling emergence rate that was higher (97.6%) than that of the control group (92%). To confirm the feasibility of the new method, DNA was extracted from plants of a BC1F2 population by two different methods, namely, from endosperm using our rapid, high-throughput method (ER-DNA) and from young leaves emerging from the same sampled seed using the CTAB method (LC-DNA). The ER-DNA was undetectable by agarose gel electrophoresis, but was found to be an adequate replacement for LC-DNA for the amplification and detection of simple sequence repeats (SSRs). Further analysis revealed that ER-DNA was generally suitable for the generation of specific 500-750-bp fragments, but not for the amplification of 1,000-2,000-bp fragments. Our rapid, high-throughput method therefore has no deleterious effects on wheat seeds and yields DNA for SSR genotyping that is a suitable alternative to traditionally obtained DNA.

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