The development and application of a Dot-ELISA assay for diagnosis of southern rice black-streaked dwarf disease in the field

Abstract

Outbreaks of the southern rice black-streaked dwarf virus (SRBSDV) have caused significant crop losses in southern China in recent years, especially in 2010. There are no effective, quick and practicable methods for the diagnosis of rice dwarf disease that can be used in the field. Traditional reverse transcription-polymerase chain reaction (RT-PCR) methodology is accurate but requires expensive reagents and instruments, as well as complex procedures that limit its applicability for field tests. To develop a sensitive and reliable assay for routine laboratory diagnosis, a rapid dot enzyme-linked immunosorbent assay (dot-ELISA) method was developed for testing rice plants infected by SRBSDV. Based on anti-SRBSDV rabbit antiserum, this new dot-ELISA was highly reliable, sensitive and specific toward SRBSDV. The accuracy of two blotting media, polyvinylidene fluoride membrane (PVDF membrane) and nitrocellulose filter membrane (NC membrane), was compared. In order to facilitate the on-site diagnosis, three county laboratories were established in Shidian (Yunnan province), Jianghua (Hunan Province) and Libo (Guizhou province). Suspected rice cases from Shidian, Yuanjiang and Malipo in Yunnan province were tested and some determined to be positive for SRBSDV by the dot-ELISA and confirmed by the One Step RT-PCR method. To date, hundreds of suspected rice samples collected from 61 districts in southwestern China have been tested, among which 55 districts were found to have rice crops infected by SRBSDV. Furthermore, the test results in the county laboratories showed that Libo, Dehong (suspected samples were sent to Shidian) and Jianghua were experiencing a current SRBSDV outbreak.

Keywords: One Step RT-PCR; SRBSDV; county laboratories; detection; dot-ELISA.

Figure 1

(A) Titers of the three SRBSDV S10 polyclonal antibodies. 1-3 represent antibodies 1, 2, 3 respectively. (B) Specificity analysis of the three SRBSDV S10 polyclonal antibodies 1, 2, 3 (from left to right).

Figure 2

(A) 1:1000-fold antiserum dilution and 60 min second blocking time. (B) 1:1500-fold antiserum dilution and 60 min second blocking time. (C) 1:1500-fold antiserum dilution and 30 min second blocking time.

Figure 3

Distribution of SRBSDV infection in China (2011). Red indicates the samples infected with SRBSDV, the blue ones represent those uninfected.

Figure 4

Positive controls were rice plants from Libo Guizhou province with confirmed SRBSDV infection. Negative controls were healthy rice plants grown in a greenhouse. (A) Dot-ELISA test (PVDF membrane) results of suspected rice plants from Shidian and Yuanjiang in Yunnan province. 1(+): Positive control. 2(+): Sample I from Shidian. 3(+): Sample II from Shidian. 4(+): Sample I from Yuanjiang. 5(+): Sample II from Yuanjiang. 6(-): Negative control. (B) Dot-ELISA test (PVDF membrane) results of suspected rice plants from Malipo Yunnan province. 1(+): Positive control. 2(+): Sample I. 3(+): Sample II. 4(+): Sample III. 5(-): Negative control. (C) One Step RT-PCR test results of suspected rice plants from Shidian and Yuanjiang. M: M, DL 500 bp DNA marker. 1: S10 gene amplification of rice sample I from Yuanjiang. 2: S10 gene amplification of rice sample IV from Shidian. 3: Reference gene amplification of rice sample I from Yuanjiang. 4: Reference gene amplification of rice sample IV from Shidian. 5: Multiple PCR of sample I from Yuanjiang. 6: Multiple PCR of sample IV from Shidian. 7: S10 gene amplification of positive sample. (D) One Step RT-PCR test results of suspected rice plants from Malipo. M: M, DL 500 bp DNA marker. 1: S10 gene amplification of rice sample I. 2: S10 gene amplification of rice sample II. 3: S10 gene amplification of rice sample III. 4: Positive control. 5: Reference gene amplification of rice sample I. 6: Reference gene amplification of rice sample II. 7: Reference gene amplification of rice sample III.

Figure 5

Comparative genomic sequence of SRBSDV between Shidian and Shaxian.

Figure 6

Dot-ELISA test (PVDF membrane) results of suspected rice plants from Yunnan province. 1(-): Sample I from Menghai. 2(-): Sample II from Menghai. 3(-): Sample III from Menghai. 4(-): Sample IV from Menghai. 5(+): Sample from Shidian. 6(-): Sample I from Lincang. 7(+): Sample II from Lincang. 8(+): Sample from Chuxiong. 9(+): Positive control. 10(-): Negative control.

Figure 7

(A) Dot-ELISA test (PVDF membrane) results from Tianzhu Guizhou Province and Liangping Chongqing City. 1(+): Positive control. 2(+): Positive control. 3(+): Sample I from Tianzhu. 4(+): Sample II from Tianzhu. 5(+): Sample III from Tianzhu. 6(+): Sample from Liangping. 7(-): Negative control (stem). 8(-): Negative control (root). (B) Dot-ELISA test (NC membrane) results from Tianzhu and Liangping. 1(+): Positive control. 2(+): Positive control. 3(+): Sample I from Tianzhu. 4(+): Sample II from Tianzhu. 5(+): Sample III from Tianzhu. 6(+): Sample from Liangping. 7(-): Negative control (root). 8(-): Negative (stem).

Figure 8

Dot-ELISA test (NC membrane) results of suspected rice plants. (A) Luodian of Guizhou Province. 1(+): Positive control. 2(+): Sample from Dongjia in Luodian. 3(+): Sample from Bianyang in Luodian. 4(-): Negative control of root. 5(-): Negative control of stem. (B) Jiajiang of Sichuan province and Dushan of Guizhou Province. 1(+): Positive control. 2(-) - 7(-): Stem of sample from Jiajiang. 8(+): Sample from Dushan. 9(-): Negative control of root. 10(-): Negative control of stem. (C) Kaiyang and Huishui of Guizhou Province. 1(+): Positive control. 2(-): Root of sample from Kaiyang. 3(-): Stem sample from Kaiyang. 4(-): Stem of sample I from Huishui. 5(-): Root of Sample I from Huishui. 6(-): Root of Sample II rom Huishui. 7(-): Stem of sample II from Huishui. 8(-): Negative control of stem. 9 (-): Negative control of root.

Figure 9

Dot-ELISA test (NC membrane) results of suspected rice plants from Yunnanand Sichuan provinces. (A) 1(+) – 7(+): Kaiyuan, Jinghong, Shizong, Yongshan, Mile, Pingbian and Mengzi respectively. 8(-): Negative control of stem. 9(+): Positive control. (B) 1(+): Positive control. 9(+): Negative control of stem. 2(+) – 10(+): Mengla, Ludian, Gejiu, Yiliang, Fengqing, Shuifu, Zhenxiong, Yanjin. (C) 1(+): Positive control. 9(+): Negative control of stem. 2(+) – 10(+): Maguan, Jiangcheng, Eshan, Kunming, Qiubei, Yuxi, Fushun, Hejiang.

Figure 10

Typical characterization of tillering stage rice plants infected with SRBSDV from three county laboratories.

Figure 11

Results in three county laboratories. (A) Libo of Guizhou province. 1(-): Negative control (stem tissue). 2(+) - 5(+): Suspected samples. 6(+): Positive control. (B) Shidian of Yunnan province. 1(+) - 6(+): Suspected samples collected from Shidian in Yunan province. 7(+): Positive control. (C) Jianghua of Hunan province. 1(+) - 5(+): Suspected samples. 6(+): Positive control. 7(-): Negative control (stem tissue).