Development of a loop-mediated isothermal amplification assay for the rapid detection of Russula subnigricans and Russula japonica

Abstract

Russula subnigricans is the only deadly species in the genus Russula with a mortality rate of more than 50%, and Russula japonica is the most common poisonous species, making rapid species identification in mushroom poisoning incidents extremely important. The main objective of this study was to develop a rapid, specific, sensitive, and simple loop-mediated isothermal amplification (LAMP) assay for the detection of R. subnigricans and R. japonica. Two sets of species-specific LAMP primers targeting internal transcribed spacer (ITS) regions were designed to identify R. subnigricans and R. japonica. The results demonstrated that while LAMP could specifically detect R. subnigricans and R. japonica, the polymerase chain reaction (PCR) could not distinguish R. subnigricans from Russula nigricans. In addition, the results demonstrated that, compared to electrophoresis-LAMP and real-time quantitative LAMP (RT-qLAMP), the detection sensitivity of HNB-LAMP (a mixture of LAMP with hydroxy naphthol blue (HNB) dye) for R. subnigricans could reach 0.5 pg/μl and was 100-fold higher than that of PCR. The LAMP reaction could be completed in 45 min, which is much faster than the conventional PCR. In the future, LAMP can be used a quick, specific, and sensitive detection tool in various fields.

Keywords: ITS; Russula japonica; Russula subnigricans; loop-mediated isothermal amplification; mushroom poisoning.

Figure 1

Basidiomata of Russula species. (A) and (B): Russula subnigricans; (C): R. nigricans; (D): R. densifolia; and (E) and (F): R. japonica.

Figure 2

Multiple-sequence alignment of internal transcribed spacer (ITS) of Russula samples. The target regions used for designing loop-mediated isothermal amplification (LAMP) primers were labeled. LAMP primers for R. subnigricans (A) and LAMP primers for R. japonica (B).

Figure 3

Schematic illustration of the LAMP assay.

Figure 4

The amplification plot and corresponding colors of the LAMP mixture over time. A sky blue color was observed in the case of amplification, whereas the negative control remained violet after the reaction. PC, positive control; NC, negative control.

Figure 5

Specificity of the polymerase chain reaction (PCR) assay for R. subnigricans. Amplification was detected by agarose gel electrophoresis of PCR amplification products. M: 100 bp ladder; lanes 1–9: R. subnigricans; lanes 10–13: R. nigricans; lane 14: R. densifolia; lanes 15–16: R. crustosa; lane 17: R. mairei; lane 18: R. pulchra; lane 19: R. chiui; lane 20: R. risigallina; lane 21: R. virescens; lane 22: R. zvarae; lane 23: R. griseocarnosa; lane 24: R. senecis; lane 25: R. sp.; lane 26: R. rosea; lane 27: Lactarius kesiyae; lane 28: R. japonica; lane 29: R. japonica; lane 30: Lactarius vividus; lane 31: R. cyanoxantha; and lane 32: ddH₂O (blank control).

Figure 6

Specificity of the LAMP assay for R. subnigricans. Hydroxy naphthol blue (HNB) dye staining of LAMP products (A) and agarose gel electrophoresis of LAMP products (B). M: 100 bp ladder; lanes 1–9: R. subnigricans; lanes 10–13: R. nigricans; lane 14: R. densifolia; lanes 15–16: R. crustosa; lane 17: R. mairei; lane 18: R. pulchra; lane 19: R. chiui; lane 20: R. risigallina; lane 21: R. virescens; lane 22: R. zvarae; lane 23: R. griseocarnosa; lane 24: R. senecis; lane 25: R. sp.; lane 26: R. rosea; lane 27: L. kesiyae; lane 28: R. japonica; lane 29: R. japonica; lane 30: L. vividus; lane 31: R. cyanoxantha; and lane 32: ddH₂O (blank control).

Figure 7

Specificity of the LAMP assay for R. japonica. HNB dye staining of LAMP products (A) and agarose gel electrophoresis of LAMP products (B). M: 100 bp ladder; lanes 1–3: R. japonica; lane 4: R. subnigricans; lane 5: R. nigricans; lane 6: R. densifolia; lane 7: R. crustosa; lane 8: R. crustosa; lane 9: R. mairei; lane 10: R. pulchra; lane 11: R. chiui; lane 12: R. risigallina; lane 13: R. virescens; lane 14: R. zvarae; lane 15: R. griseocarnosa; lane 16: R. senecis; lane 17: R. sp.; lane 18: R. rosea; lane 19: R. cyanoxantha; and lane 20: ddH₂O (blank control).

Figure 8

Sensitivity of the PCR assay for R. subnigricans. A dilution series of R. subnigricans DNA was prepared as follows: (1) 5 ng; (2) 500 pg; (3) 50 pg; (4) 5 pg; (5) 500 fg; (6) 50 fg; and (7) no template for negative control.

Figure 9

Sensitivity of the LAMP assay for R. subnigricans. A dilution series of R. subnigricans DNA was prepared as follows: (1) 5 ng; (2), 500 pg; (3) 50 pg; (4) 5 pg; (5) 500 fg; (6) 50 fg; and (7) no template for negative control. (A) Real-time quantitative LAMP (RT-qLAMP) analysis with different DNA concentrations. The standard curve based on this dilution series showed a linear relationship between the log of the quantity of initial template DNA (lg conc.) and threshold time (Tt). The coefficient of determination (R²) of the linear regression was 0.9802. (B) Electrophoresis-LAMP with different DNA concentrations. (C) HNB-LAMP with different DNA concentrations.