Dot immunobinding assay method with chlorophyll removal for the detection of southern rice black-streaked dwarf virus

Abstract

Southern rice black-streaked dwarf virus (SRBSDV), a new virus from Fiji, has seriously damaged rice crops in southern China and northern Vietnam in recent years. This virus is difficult to diagnose in the early stages of infection, and is very destructive at the late stage. In the present study, a dot immunobinding assay (DIBA) that has a high sensitivity for diagnosing SRBSDV was developed. Two kinds of treatment for the DIBA were evaluated to determine the most effective one for removing chlorophyll interferences via rice extraction. The first included several reagents to remove chlorophyll, namely, the alkaline reagents like magnesium oxide and alumina oxide, the adsorbent reagents like activated carbon and bentonite, as well as the extraction agent acetone. The second and third treatments, which were used to remove chlorophyll in blot membrane-nitrocellulose and polyvinylidene fluoride (PVDF), included several organic solvents containing methanol, ethanol, acetone, ethyl acetate, and diethyl ether. The results showed that activated carbon and methanol yielded the best contrasting purple color for the infected samples by decreasing the chlorophyll content.

Figure 1

Effect of removing chlorophyll from each reagent treatment before and after centrifugation. (A) 1–4 represent magnesium oxide, alumina oxide, bentonite, and activated carbon (50, 100, and 150 mg/mL) added to the rice extract in carbonate buffer in Eppendorf tubes shaken for 3 min;5 is rice extract control in carbonate buffer without a reagent-removing chlorophyll. (B) 1–5 represent the same above listed treatment groups, which were centrifuged at 12,000 × g and 4 °C for 10 min.

Figure 2

Determination of chlorophyll content in several reagents. Using the SPSS 11.5 software [24], data were statistically analyzed byANOVA (least significant difference). The results showed differences between the statistical data of the carbonate buffer treatmentand magnesium oxide, alumina oxide, bentonite, or activated carbon at 150 mg/mL (p < 0.05). There were statistically significant differences between the carbonate buffer treatment and magnesium oxide or bentonite at 50 and 100 mg/mL (p < 0.05). There were statistically significant differences in the same treatment group of magnesium oxide, alumina oxide, and activated carbon at 50 and 100 mg/mL (p < 0.05).

Figure 3

Color of NC membrane after DIBA processing with TBST and 5% non-fat dry milk for blocking as well as antibody and chromogenic detection of alkaline-phosphatase. Numbers 1–4 respectively represent magnesium oxide, alumina oxide, bentonite, and active carbon at 50, 100, and 150 mg/mL, respectively. Number 5 represents the rice extract in carbonate buffer, and number 6 represents the rice extract in buffer after acetone precipitation. Letters A and B represent two repeated samples. The rice infected by the SRBSDV was from the rice sample cultivated in the greenhouse.

Figure 4

Effect of rice extract loading onto NC and PVDF membranes before and after washing. Numbers 1–3 represent the dry effect after loading the rice extract on the NC membrane. Numbers 4–6 represent the dry effect after loading the rice extract onto PVDF. Numbers 7–11 represent the washing effect of five kinds of organic solvent. A–B represent two repeats of the same experimental sample.

Figure 5

Determination of chlorophyll content in the PVDF membrane by washing with organic solvent. The chlorophyll content was presented as mean ± SD. Using SPSS 11.5, the data was statistically analyzed byANOVA (least significant difference). The results showed differences between the statistical data of the control and other treatment groups (p < 0.05), between methanol and ethyl acetate (p < 0.05),between ethanol and ethyl acetate (p < 0.05), between acetone and diethyl ether(p < 0.05), as well as between ethyl acetate and diethyl ether(p < 0.05).

Figure 6

DIBA test completed in the PVDF membrane. (A) The dry effect of rice extraction in carbonate-coating buffer loading onto the PVDF membrane. (B) The effect of washing the membrane by methanol. (C) The coloration effect of the PVDF membrane with nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-inodlyl-phosphate (BCIP). Numbers 1 and 2 represent SRBSDV-infected rice in the greenhouse. Numbers 3 and 4 represent SRBSDV-infected rice acquired from Yuanjiang, Yunnan in 2011. Numbers 5 and 6 represent SRBSDV-infected rice acquired from Shidian, Yunnan in 2011. Numbers 7 and 8 represent SRBSDV-infected rice acquired from Dushang, Guizhou in 2010.

Figure 7

Demonstration test of IC-RT-PCR against a positive blot in the DIBA assay around the PVDF. “M₁” and “M₂” represent the DNA molecular weight marker (DL500 and DL1000 Marker, TaKaRa, Dalian). Part A: Lane 1 and 2 represent the gene product of S5 and S10. Part B and C: Lanes “1–3”, “4–6” and “7–9” represent the SRBSDV-infected rice from the greenhouse at three dilution folds: 10×, 100×, and 1,000×.

Figure 7

Treatments for removing chlorophyll from rice extract solutions.