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Title: One-step reverse transcription loop mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus

Author
item PENG, DANDAN - China Agricultural University
item XLE, JIPENG - China Agricultural University
item QIANG, WEI - China Agricultural University
item Ling, Kai-Shu
item GUI, LIYUN - China Agricultural University
item FAN, ZAIFENG - China Agricultural University
item ZHOU, TAO - China Agricultural University

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/9/2017
Publication Date: 7/15/2017
Citation: Peng, D., Xle, J., Qiang, W., Ling, K., Gui, L., Fan, Z., Zhou, T. 2017. One-step reverse transcription loop mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus. Journal of Virological Methods. 248:154-158.

Interpretive Summary: Apple chlorotic leaf spot virus (ACLSV) infects many fruit trees, including apple, peach, pear, plum, quince, cherry and apricot. It was first discovered on apple trees in 1959 and now it is widely distribution around the world. The virus is transmitted mechanically and by grafting. ACLSV induces latent infection on most host plants, causing no visible symptoms on leaves and fruits in the infected trees. Therefore, it is difficult to monitor or identify based on symptom observation in the field. Currently, laboratory assays are available to detect ACLSV. However, as the virus has a very low titer in infected trees, false negative is quite common. Moreover, many disease diagnostic clinics in developing countries may not be equipped with sophisticated molecular biological capabilities. Hence, developing a sensitive, field-oriented detection method that would offer a reliable and rapid disease diagnosis is of particular interest to fruit growers. Loop-mediated isothermal amplification (LAMP) is a method capable of amplifying the virus molecule under isothermal conditions with high specificity and efficiency. This LAMP technology has been successfully applied to detect various plant pathogens. In this study, a LAMP assay was developed to allow for sensitive detection of ACLSV. The detection requires only a heated water bath, and therefore can be easily performed under simple field conditions, particularly in many developing countries. In light of this simplicity and cost efficiency, this technique is suitable for rapid detection of ACLSV infected trees. Thus, an appropriate management tactic could be taken to prevent its further spread.

Technical Abstract: A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Apple chlorotic leaf spot virus (ACLSV) was developed. In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and was synthesized for the RT-LAMP assay using total RNA extracted from ACLSV-infected leaf tissues. The optimal reaction temperature and assay time were determined to be 64 C for 75 min. The sensitivity of RT-LAMP reactions reached to the dilution of 1:3125, which was more sensitive than RT-PCR assay. The successful application of RT-LAMP to field-collected apple samples demonstrated its potential broader applications for effective disease diagnosis, thus to control ACLSV from further spread, particularly in many developing countries around the world. To our knowledge, this is the first application of RT-LAMP for detection of ACLSV.