**2.6 Designing of primers**

Sets of 3 primers (**Table 1**); elongation factor 1 alpha (*Ef1ɑ*), *β*-tubulin, and membrane protein (*MEP*) were used in the current research. *MEP* and *Ef1ɑ* were


**Table 1.** *Specific primers used in the current study.*

**515**

*Fungal Biomass Load and* Aspergillus flavus *in a Controlled Environment*

obtained from Dr. Shane Murray (pers. comm) whereas, *β-*tubulin was designed in Primer3 ver. 4.0 programme [19]. Secondary structure formation was evaluated in DNAMAN software ver. 6.0 (Lynnon LLC., USA) and further verified in OligoAnalyzer Tool (Integrated DNA Technologies). The melt curve and PCR analysis were used to identify both non-specific and specific amplification.

Conventional polymerase chain reaction amplification was carried out in volumes of 25 μl and consisted of 0.5 μl of 10 μM dNTPs (Bioline), 10× reaction buffer with MgCl2, 1 μl of 10 μM forward and reverse primers, 0.2 μl Kapa Taq, 1 μl of 10 ng DNA template, and sterile H20. Protocol performed and followed for cycling conditions were: 1 cycle for 5 min at 94°C followed by 35× (for 30 s at 94°C, for 45 s at 60°C, for 90 s at 72°C). Elongation step was achieved at 72°C for 7 min and finally at 4°C for 1 min. The products of PCR were assessed on 2% agarose/EtBr gel in TAE1 X buffer (Tris–acetate 40 mM and EDTA 1.0 mM). Fermentas (100 bp

Ten-fold serial dilutions of pooled 10 ng gDNA extracts from *A. flavus* and control plants were used to create standard curves. The threshold cycle (Ct) values for each dilution were plotted against the logarithm of the starting quantity of the template. The amplification efficiencies were created from the std. curve slopes according to the methods [13, 20]. Additionally, linear regression curves were

The quantity of targeted DNA in an unknown sample was inferred from the

Ten nanograms of DNA isolated from infected and healthy maize roots and shoots respectively were used to assess primer specificity. For the exclusion of false negative results, template DNA samples from fungi were assessed for polymerase chain reaction amplification with primer pairs *EF1ɑ* and *β*-Tub. DNA extracted from pure fungal cultures (*A. flavus*) and control plant tissues were pooled, diluted to 10 ng/μl and used to evaluate the quantity of fungal DNA template in the infected

plant tissue. The final fungal DNA template concentrations were 1, 5 × 10−1, 2.5 × 10−1, 1.25 × 10−1, 6.25 × 10−2, 3.125 × 10−2 ng/μl. These dilutions were used to estimate the detection limits of the *EF1ɑ* and β-Tub primer pair in the infected plant tissues. Serial dilutions of extracted DNA from healthy maize tissue were prepared to gauge the detection limits of the *MEP*. For normalization and quantification of gene between different samples, the amount of fungal DNA as calculated by the *Ct* value for *β-*Tub and/or *EF1ɑ* was divided by the amount of maize DNA as calculated by the *Ct* values for MEP. Rotor Gene 6000 2 plex HRM (Corbett Life Science Research, Australia) was used to assess the profiles of gene expression. Kapa *SYBR* Fast Kit, Master mix (Kapa BioSystems, South Africa) containing DNA polymerase, reaction buffers, dNTPs and 3 mM MgCl2 were used for each polymerase chain reaction. Final concentrations of 10 μM gene specific primers (0.4 μl reverse and 0.2 μl forward), 1× Kapa SYBR green and 1 μl gDNA template were prepared to 20 μl total volume using nuclease free H2O. Primer sets of specific genes (**Table 1**)

were used in separate reactions which were performed in triplicate.

The quality and integrity of the isolated DNA, samples from infected and control tissues of the maize, and saprophytic fungi were subjected to polymerase chain reaction analysis with the reference genes under the following amplification

*Slope* <sup>−</sup> <sup>=</sup>

.

*DOI: http://dx.doi.org/10.5772/intechopen.93307*

DNA ladder) was used as a molecular size marker.

**2.8 Standard curves and fungal quantification**

drawn, and the qPCR efficiency was calculated as: <sup>1</sup> *<sup>E</sup>* <sup>10</sup>

**2.7 PCR amplification**

respective std. curves.

obtained from Dr. Shane Murray (pers. comm) whereas, *β-*tubulin was designed in Primer3 ver. 4.0 programme [19]. Secondary structure formation was evaluated in DNAMAN software ver. 6.0 (Lynnon LLC., USA) and further verified in OligoAnalyzer Tool (Integrated DNA Technologies). The melt curve and PCR analysis were used to identify both non-specific and specific amplification.
