ABSTRACT
The harm of plant virus disease is serious, which significantly restricts the sustainable development of agriculture and can cause huge economic losses. Monitoring plant health and early detection of viral pathogens are essential to reduce the spread of disease. Therefore, in order to realize the early detection of plant viral diseases in the field, a sensitive, specific and efficient colorimetric visual technique for plant viral RNA was designed by combining the colorimetric method based on gold nanoparticles (AuNPs) and hybrid chain reaction (HCR). In this study, tobacco mosaic virus (TMV) was used as a model to design two hairpin structures H1/ H2 with single-stranded tails based on TMV-specific conserved fragments. TMV could open the hairpin structure to alternately form long double-straight DNA. The binding difference between AuNPs and two nucleic acid states before and after HCR reaction resulted in colorimetric signal generation, thus realizing visual detection of TMV. After optimizing the concentration of Tris-HAc, the concentration of hairpin structure and the reaction time of HCR, the best detection conditions were obtained. The sensitivity and specificity of the technique were analyzed and real samples were tested under optimal conditions. The results showed that the absorbance ratio of AuNPs (A
ABSTRACT
Soybean mosaic virus (SMV) is a critical pathogen that reduces the soybean yield and seed quality worldwide, and SMV has a restricted natural host range. In this study, we used sequence-independent amplification (SIA) methods to identify the viruses that may cause the Atractylodes macrocephala Koidz disease. Results revealed that there is SMV in diseased Atractylodes macrocephala leaves, and we named this isolate as SMV-Am. To further characterize the genomic structural and phylogenetic relationships of SMV-Am, genomic dsRNA was extracted, and the genomic sequence was amplified by RT-PCR and RACE. The results of bioinformatics analysis showed that the genomic RNA of SMV-Am is 9587 nucleotides in length, which conforms to the typical characteristics of Potyvirus. According to the sequence alignment of complete nucleotide sequences, SMV-Am showed the highest level of nucleotide homology and amino acid sequences to SMV-Liaoning, 96. 57% and 98. 86%, respectively. In addition, phylogenetic analysis based on the nucleotide sequences of other SMV isolates of SMV-Am revealed that SMV-Am was most closely related to SMV-Liaoning. Then, the SMV-Am protein was further analyzed by I-TASSER and PyMOL software, revealing that the key amino acid mutations led to the structural changes of P1, HC-Pro, P3, 6K2, NIa-pro and NIb proteins, with P1 the most obvious. Finally, recombination has been detected at the position of 6560-8950 nucleotides, the main parent is the isolate SMV-XFQ012 (accession number KP710875. 1), and the secondary parent is isolate SMV-pCB301-SC15 (accession number MH919386. 1). This study is the first report of SMV in Atractylodes macrocephala Koidz, and these results are expected to provide a scientific basis for the prevention and control of SMV infection on Atractylodes macrocephala Koidz.
ABSTRACT
Plant virus diseases are one of the major diseases restricting erop production.Timely identification of their pathogen and development rules is the prerequisite for effective control of their large- scale spread.However, long cycle, tedious steps and strict detection environment were the disadvantages existing in the detection technology of plant virus disease.In this study, Tobacco Mosaic virus (TMV) was used as a model to he extract UNA based on CMBs-ACPtmv , which was design based on the principle of complementary base pairing.Meanwhile, the experimental conditions were optimized and analyzed, including the preparation conditions of functionalized magnetic beads, the reaction conditions during extraction, and the sensitivity, stability and other properties of the method.The results showed the ability to capture RNA of CMBs-ACPtmv were best when prepared with 4 fxmol capture probe (ACPTMV ) and 0.08 mg carboxyl magnetic beads (CMBs) ; After 3 min of extraction, CMBs-ACPtmv has the best RNA extraction effect, but when the extraction temperature of CMBs-ACPtmv was changed, its extraction capacity showed no significant change; In the comprehensive performance evaluation, the sensitivity of CMBs-ACPjjjv can reach 2.5 ng/fxL, and the detection stability is good.Compared with conventional RNA extraction technology, CMBs-ACPimv has outstanding advantages in detection time and sample consumption.The functional magnetic beads extraction method established in this study is fast, safe and simple.It can achieve rapid extraction of plant virus RNA with simple equipment, which has a broad application prospect.