Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification

Abstract

For the detection of wheat yellow mosaic virus (WYMV), we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Using Primer Explorer software, four sets of primers were designed and RT-LAMP assay reaction conditions were optimized. The RT-LAMP was performed at different times by four primer sets. Agarose gel analysis showed that WYMV could be detected after 30 min with the primer set III and after 45 min with the other three primer sets, both under the 80-min reaction time. RT-LAMP had the same results with the four primer sets, thus primer set III and 65°C for 80 min reaction were selected for virus detection. There was no significant different when avian myeloblastosis virus (AMV) and moloney murine leukemia virus (M-MLV) RT-LAMP with the four primer sets and M-MLV was chosen due to its relatively cheap price. The result on specificity showed that the assay could amplify WYMV specifically, and the sensitivity comparison showed that the RT-LAMP was 100 times more sensitive than conventional reverse-transcriptase-polymerase chain reaction (RT-PCR). Overall, RT-LAMP was found to be a simple, specific, sensitive, convenient and time-saving method for WYMV detection.

Figure 1

Primer locations. Four sets of primers were designed, named I-IV, which targeted the CP (RNA1) gene and 72 kDa (RNA2) gene of WYMV. HC72-157 F and HC72-518R were the conventional RT-PCR primers

Figure 2

Agarose gel analysis of RT-LAMP with four primers sets at different reaction times. Total RNA was extracted from WYMV-infected wheat leaves and detected by RT-LAMP. I-IV were four sets of primers; M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: positive controls

Figure 3

Agarose gel analysis of different RT-LAMP reactions. I-IV were four sets of primers; AMV was AMV-mediated RT-LAMP; M-MLV was M-MLV-mediated RT-LAMP. M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: virus-infected samples

Figure 4

RT-LAMP reaction mixture turbidity (left) and agarose gel analysis (right). I-IV were four sets of primers. M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: WYMV-infected samples

Figure 5

Specificity of primers for WYMV-infected samples by agarose gel analysis. (a) and (b) were RT-LAMP; (c) and (d) were RT-PCR for WYMV; (e) was RT-PCR for CWMV; (f) was RT-PCR for BSMV. The arrows showed the CWMV-positive band (left) and BSMV-positive band (right). M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: CWMV-infected wheat leaves (left) and BSMV-infected wheat leaves (right); 5 and 6: WYMV-infected wheat leaves

Figure 6

Comparison of detection sensitivity between RT-LAMP and RT-PCR for WYMV. Total RNA extract was serially diluted in 10-fold increments (10⁰ to 10^-8) with healthy wheat RNAs. The arrow showed the positive band by RT-PCR. M: 100-bp DNA ladder marker; 1-9: total RNA dilution (10⁰ to 10^-8); 10: healthy control

Figure 7

Detection of field samples by RT-LAMP (a) and RT-PCR (b). M: 100-bp DNA ladder marker; 1-12: wheat samples collected from Yushan and Xiaqiao, Jiangsu Province: 11-6982, 11-6984, 11-6987, 11-7081, 11-7082, 11-7083, 11-7000, 11-7084, 11-7085,11-7086, 11-7087 and 11-7090, respectively; 13: healthy control; 14: positive control