Establishment And Application Of Nucleic Acid Spot Hybridization And Quantitative Real-time PCR Used For Detection Of Mulberry Crinkle Leaf Virus

Mulberry is a perennial woody plant of the genus Morus,which has a long history of cultivation in China.Mulberry leaves are not only an important raw material for sericulture,but also a traditional Chinese medicine with effects of anticoagulant and lowering blood pressure.Mulberry trees are susceptible to pathogens and disease under natural growth.Mulberry crinkle leaf virus(MCLV)is a new geminivirus reported in recent years.It has been reported in many silkworm areas in China.Its damage to mulberry trees cannot be assessed because the relationship between mulberry crinkle leaf virus and mulberry disease has not been determined by Koch's postulates.Additionally,Due to the lack of effective chemical agents for the prevention and control of plant viruses,early diagnosis and early treatment are effective methods for preventing viral diseases from spreading through seedlings.Therefore,the establishment of an efficient,highly sensitive and simple detection system can effectively prevent the spread of MCLV.The research contents in this paper include the establishment and application of nucleic acid spot hybridization(NASH)method for detection of mulberry crinkle virus,and the establishment and application of Quantitative Real-time PCR(qPCR)for detection of MCLV.1.Establishment and Application of Nucleic Acid Spot Hybridization Method Used For Detection of Mulberry Crinkle Leaf Virus:DNA probes specific and sensitive to mulberry crinkle leaf virus were labeled with non-radioactive digoxigenin(DIG)and were generated according to the random-primed DNA labeling method.A nucleic acid spot hybridization(NASH)method was developed using the DIG-labeled DNA probes to detect mulberry crinkle leaf virus(MCLV)by optimizing the hybridization time and loading volumes.The diagnosis accurate rate of the established MCLV-specific NASH was 93.1% compared to PCR.Sixty samples grown in three different mulberry fields(A,B,C)were collected and detected by NASH to investigate the distribution of MCLV in this area and the relationship between the MCLV and mulberry disease with virus-like symptoms.The results showed that 4 out of 20 samples(20%)in field A,18 out of 20 samples(90%)in field B,and 19 out of 20 samples(95%)in field C were detected to be infected with MCLV.The average infection rate was approximate 68.3%,indicating that MCLV is widely distribution in this area.The detected results also showed that no MCLV was detected in leaf samples from the mulberry trees with obvious mosaic or(and)dwarf symptoms,while MCLV was detected in leaf samples from the mulberry trees without any virus-like symptoms,suggesting that MCLV might not be responsible for mosaic or dwarf symptoms exhibiting on the mulberry trees.2.Establishment and application of Quantitative Real-time PCR(qPCR)for mulberry crinkle leaf virus: Specific primers were designed based on nucleotide acid(nt)sequence of MCLV coat protein.The full length 738 nt sequence of MCLV cp gene was obtained by PCR using total DNA extracted from MCLV-positive mulberry leaves as template,cloned,and sequenced.The results showed that the obtained sequence was identical to that of the MCLV cp gene deposited in GenBank(KR131749).Three pairs of primers for qPCR were designed based on the obtained cp gene sequence.The specific results of PCR using the recombinant plasmid containing the full-length sequence of cp gene as a template showed that two of three primers were suitable to be used for qPCR.The recombinant plasmid containing the full-length sequence of the cp gene was used as standard sample,and was subjected to 10-fold serial dilution.SYBR Green I qPCR using the dilution samples was performed to establish the standard curve.The results showed that the linear regression equation of the standard curve constructed by one primer was y=-3.433x+12.925,the amplification efficiency was 95.6%,and the correlation coefficient was 0.998.The melting curve had single peak,suggesting that there was no primer dimer or non-specific amplification products,and that the primer was specific to MCLV.Additionally,qPCR was sensitive to detect MCLV(? 47.8 copies/?l),and the variation coefficient of each dilution sample was less than 1.56%,suggesting that the standard curve is credible,the primer is specific,the detection sensitivity is high,and the repeatability is good.The another primer was not suitable for qPCR because the constructed standard curve could not establish a linear relationship.The detection results of samples collected from mulberry fields using the established qPCR showed that all 15 samples were positive for MCLV.The result was consistent with that of routine PCR.qPCR is not only used for the detection of a large number of samples collected from the fields,but also for quantitative analysis of MCLV in samples,which is more advantageous than PCR detection.