Rapid and Sensitive Detection of Meloidogyne graminicola in Soil Using Conventional PCR, Loop-Mediated Isothermal Amplification, and Real-Time PCR Methods.

Meloidogyne graminicola is one of the major plant-parasitic nematodes (PPNs) that affect rice agriculture. Rapid identification and quantification of M. graminicola in soil is crucial for early diagnosis so that measures can be taken to reduce the impact of PPN diseases and ensure food security. In this study, M. graminicola species-specific primers for conventional PCR, loop-mediated isothermal amplification (LAMP), and real-time PCR were designed based on the sequence-characterized amplified region. The primers were highly specific and sensitive, and only samples containing M. graminicola DNA showed positive results. The sensitivity of LAMP and real-time PCR (two second-stage juvenile [J2] M. graminicola in 100 g of soil) was higher than that of conventional PCR (200 J2s in 100 g of soil). A standard curve (correlation coefficient R2 = 0.970, P < 0.001) was generated by amplifying DNA extracted from 0.5 g of soil, and a significant correlation was observed between the number of M. graminicola determined by microscopic examination and that predicted from the standard curve (R2 = 0.477, P = 0.0160). In quantification analyses of M. graminicola isolated from 31 naturally infested soils, the sensitivity of LAMP and real-time PCR (22 M. graminicola in 100 g of soil) was higher than that of conventional PCR (211 M. graminicola in 100 g of soil). The conventional PCR, LAMP, and real-time PCR methods have the potential to provide a useful platform for rapid species identification according to the experimental conditions. The real-time PCR assay and standard curve can be used for quantification of M. graminicola. These newly developed assays will help to facilitate the control of these economically important PPNs.

Selection of reliable reference genes for gene expression studies in Caenorhabditis elegans exposed to crystals (Cry1Ia36) protein of Bacillus thuringiensis.

Quantitative real time PCR (qRT-PCR) is a nucleic acid quantitative technique and is also considered as a validation tool. The Cry1Ia36 protein isolated from Bacillus thuringiensis (Bt) strain YC-10 has high nematicidal activity against nematodes. Caenorhabditis elegans is one of the major model organisms and a readily accessible source of biological material for gene expression studies. To evaluate the expression stability of 12 candidate reference genes of C. elegans for exposing to different concentrations of Cry1Ia36 protein and different treat time, five statistical approaches (the comparative delta-Ct method, BestKeeper, NormFinder, Genorm and RefFinder) were used to evaluate each individual candidate reference gene. The results indicated that cdc-42 and F35G12.2 were the best reference genes for performing reliable gene expression normalization in the impact of Cry1Ia36 protein. In addition, when C. elegans was exposed to Cry1Ia36 protein and other nematicides, avermectin and 5-aminolevulinic acid, cdc-42 was recommended as the most reliable reference genes. Y45F10D.4 was the least stable reference genes in our experimental settings. Therefore, cdc-42 was reliable reference gene for gene expression studies in C. elegans exposed to Cry1Ia36 protein and other nematicides.