A TaqMan-based Quantitative RT-PCR Method for Detection of Apple Chlorotic Leaf Spot Virus in Hawthorns

ACLSV is the type species of genus Trichovirus in family Betaflexiviridae [1]. ACLSV causes a severe decline in trees and the quality of fruits, is transmitted by the grafting, pruning, or propagation of materials and nematodes, and has an extensive host range of most fruit tree species of family Rosaceae, including apple, pear, peach, plum, almond, apricot, cherry and hawthorn [2]. The virus genome is composed of 7474–7561 nucleotides, excluding poly-adenylated tails. It contains three overlapping open reading frames (ORFs) that encode a 216-kDa replication-associated protein (Rep), a 50-kDa movement protein (MP), and a 22-kDa coat protein (CP) [3-4]. 

The coat protein is the only constitutive protein, although this region is the most conserved part of the genome, sequence diversity has been reported among many isolates [5-6]. Different isolates have great genetic diversity and molecular variation of ACLSV in China and the world. A recent study showed that ACLSV variants in the world clustered separately were unrelated to host species or geographic origin, while natural recombinant events among isolates/genotypes play a role in ACLSV evolution [7]. Recently, mixed infections by ACLSV and Apple stem grooving virus (ASGV) and/or Apple stem pitting virus (ASPV) in the same tree have been reported [8]. These viruses are transmitted with infected propagating materials. Planting virus-free propagating materials is a main method to prevent and cure virus disease, while, it first demands an accurate, rapid and reliable virus detection method. 

Conventional virus detection methods of ACLSV are based on virus isolation and serological tests, both of which are time consuming and laborious [9]. Nucleic acid based RT-PCR has been recommended for detection ACLSV because of its sensitivity, specificity and less time consuming, however the sensitive of this method remains a question when virus titer is low in plant samples and it is prone to sample contamination occurring in gel electrophoresis, which increases incidence of false-positive results [10-12]. More recently, a TaqMan-based quantitative RT-PCR method is established that allows accurate and reproducible quantitation for the detection of fruit trees viruses. The quantitative RT-PCR improves diagnosis sensitivity (10-1000 fold more sensitive than PCR/RT-PCR), practicality, reduces risk of carry-over contaminations and can quantitate estimation of viral load [13]. 

In this study, we proved the quantitative RT-PCR method developed here is a highly efficient and practical method for the detection of ACLSV in hawthorn. Since viral genomes have a relatively high mutation rate and even few nucleotide mismatches between the target sequences and the primers or the probe may lead to false negative results, therefore, the TaqMan primers and probe used in in our research were designed to fit the most conservative CP genome sequence. Standard curve from a plasmid that contained a CP gene sequence of ACLSV were used to obtain an absolute quantitation of ACLSV. The sensitivity and reproducibility of this method were investigated. In addition, this is first report on the quantitative RTPCR method that applied to detect ACLSV and to evaluate the virus present in hawthorn samples.