PCR Summary Paper

Scientists determined a nematode called Rotylenchulus reiformimis is in tropical, southern temperate and subtropical zone soils in greater than 38 states around the globe. This phytopathogenic nematode has a host range of seventy seven families with more than 300 plant species. In 2010, this reniform nematode, a foremost cotton problem in the southern United States, caused a 1.48% estimated loss in the produce of the complete US yield. The pathogen is increasing in population density and enlarging its populace radius in colonized areas.
The reniform nematode is the most damaging cotton pathogen in the majority of southern United States, exceeding the root-knot (Meloidogyne incognita). The pathogen’s distribution varies across the southern United States and in the same locations over time. The nematode has no recorded genetic resistance in the commercial upland cotton varieties though there are underway efforts to develop resistant hybrids. Present control methods rely on growing cotton in cycle with hosts that don’t harbor the host, with tolerant varieties or by application of nematicides. Another control method in for this pathogen exploration is transgenic resistance.
Reniform nematodes management depends on detection and quantification of the nematodes in advance of planting. With early identification and quantification, growers can select appropriate sequences of rotation or apply nematicides. Farmers need an exact pathogen population estimation to make decisions for crop planting. Many private and public diagnostic clinics get much soil samples from farmers who need correct identification and quantification of ranitiform and other nematode population densities in fall-collected soil specimens.
Correct diagnostic methods, however, depend on nematode extraction from soil samples physically, combined with observation and counting on light microscopy. This process requires a lot of time and specialisation. This method also takes time to report to growers as they are overwhelmed with many samples as most diagnostic samples will fall instantly after harvest.
The methodology pertains using DNA-based techniques that include: the use of PCR primer design, DNA extraction, real-time PCR, and conventional PCR. On PCR primer design, all probe and primer combinations were affirmed to contain low homodimer and heterodimer potential using the Oligoanalyser 3.1 software. The rapid DNA extraction procedure used low-cost reagents and was faster than the worm lysis buffer. All the native soil samples with reniform nematodes by inspection microscopically were also positive for the pathogen in conventional PCR. On real-time PCR, the reniform nematodes were precisely quantified over a 5, 10, 25, and 50 series of individuals using the Qiamp DNA sample. The number of reniform nematodes and cycle threshold value had a correlation of R2=0.9557.

The PCR technique, detailed procedure, and programming.

Materials

The scientists obtained reniform nematode DNA population for conventional PCR and real-time PCR assays development, from Arkansas cotton fields. Other species of nematodes used in the study include Heterodera glycines and Meloidogyne incognita that were obtained from soybean fields and also from Arkansas cotton fields.

PCR Primer Design

Researchers designed reniform nematode specific probe and primers manually by aligning many internal transcribed spacer (ITS) sequences they acquired from more than 50 reniform nematode accessions. These nematode accessions were from China, Japan, United States and Malaysia through GenBank. The scientists also included ITS sequences of Globodera pallida and Scutellonema truncatum in alignment to select regions unique to reniform nematodes.
They used these sequences because they are the most similar to the ITS sequences of reniform nematodes. Scientists then manually designed probes and primers specific to the ITS sequences of all reniform nematodes, but different from genera of related nematodes using ClustalW. The researchers then searched all primer sequences with blastn to assure specificity of ITS sequences of all reniform nematodes in the GenBank. They verified all probe and primer combinations to have low homodimer and heterodimer potential using the Oligoanalyser 3.1 software.

DNA Extraction

Scientists isolated nematodes using sugar centrifugal floatation and Baermann funnel. DNA was then extracted from many and individual nematodes for all real-time PCR, using the QIAamp DNA Micro Kit. They then used DNA from single reniform nematodes from various geographic locations as they had previously extracted primer specificity testing, using the Sigma REDExtract-N-Amp Tissue PCR Kit. They then extracted DNA for multiplex assays from nematodes extracted from soil using either the rapid DNA extraction buffer or the worm lysis buffer.
In rapid DNA extraction procedure, the researchers removed a 50 microliters bulk nematode aliquot from the bottom of the nematode sample in water, separated from the soil as explained above. They then added 25 microliters of buffer A to these nematodes and heated the mixture for 10 minutes to 95°C. Each extraction made buffer A fresh, and it contained 2% Tween 20 and 0.1 M sodium hydroxide. After this heating stage, they added 50 microliters of buffer B that consisted 2 mm EDTA and 0.1 M TrisHCL to neutralize the DNA solution extracted.
To determine the presence, in soil, of the reniform nematodes, the researchers isolated bulk nematodes using sugar centrifugal floatation and Baermann funnel. For native lands, they then isolated nematodes as described above and extracted DNA from a sample of 50 microliter bulk nematode in water using the rapid DNA extraction technique.

Conventional PCR

Scientists performed all reactions for conventional PCR using the following conditions and reagents: 200 micromoles dNTP, one unit of Taq polymerase, five times GoTaq Green Reaction Buffer, and 0.25 micromoles of reverse and forward primers. They then set the cycling conditions for Ren240 primers at 95°C for ten minutes which was followed by 35 cycles for 15 seconds at 90°C, 30 seconds at 60°C, and 60 seconds at 72°C. They the performed multiplex reactions using universal nematode primers under the same conditions, though they used 55°C for annealing rather than 60°C.

Real-time PCR

The researchers performed real-time PCR using Quantifast SYBR green two times master mix, 96-well low profile plates, and 0.25 micromoles primer concentrations. They mixed these reagents on an MX3000P qPCR thermocycler. They adjusted the cycling conditions for SYBR green with Ren240 primers as follows: 10 minutes at 95°C 15 seconds of 40 cycles at 95°C, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds.
The scientists performed TaqMan PCR using 200 microliters dNTP, reverse (Ren240R) primers, standard magnesium chloride containing ten times PCR buffer, and 0.25 micromoles forward (Ren240F) primers. The conditions for TaqMan PCR cycling were similar to the conditions for the SYBR Green except for annealing that they set at 55°C for 30 seconds. They performed all assays using three technical replications and repeated all the experiments.
The researchers constructed standard curves of recorded DNA concentrations of reniform nematode between the log DNA level and the cycle threshold (Ct). They then measured the DNA concentration of the template using a NanoDrop spectrophotometer at 260 nm.they extracted nematode DNA for all Q-PCR reactions using the Qiamp DNA Micro Kit, where DNA was eluted from the column using two microliters of sample and 50 microliters of elution buffers.

Work cited

Sayler, Ronald J., et al. "Conventional PCR detection and real-time PCR quantification of reniform nematodes." Plant Disease 96.12 (2012): 1757-1762.