Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén)

Abstract

BackgroundThe small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples.ResultsWe established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 103 fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation.ConclusionsA simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0732-6) contains supplementary material, which is available to authorized users.

Triazophos‐induced vertical transmission of rice stripe virus is associated with host vitellogenin in the small brown planthopper Laodelphax striatellus

“…Each fertilized female was subsequently labeled and raised separately for oviposition. After females died, they were selected for RSV detection via the dot immunobinding assay (DIBA) . If a female was viruliferous, its offspring were also considered viruliferous for continuous breeding.…”

“…After 2 days, the male was removed and the fertilized female was reared on rice seedlings for oviposition. Females were used for RSV detection via the DIBA method after they died. Approximately 24 h post hatching, nymphs were separately reared on fresh rice seedlings.…”

Determination of Suitable RT-qPCR Reference Genes for Studies of Gene Functions in Laodelphax striatellus (Fallén)

“…Accurate RT-qPCR results depend on several key factors, including data normalization using reliable reference gene(s). Although RT-qPCR has been used to study gene expression and functions in L. striatellus in many laboratories [2,6,15,31,32], the expression of the reference genes used in these studies was not characterized in different L. striatellus tissues or in L. striatellus under various conditions. Because the same reference genes in different insect species or in the same insect species but under different growth or environmental conditions can vary significantly, we consider that the suitability of the reported L. striatellus reference genes should be re-evaluated under different defined experimental conditions.…”

“…Non-viruliferous (NV) and RSV viruliferous L. striatellus was originally collected from Haian, in the Jiangsu Province, China, and maintained in the laboratory as described previously [2]. The insects were reared on rice seedlings inside a chamber set at 25 ± 3 °C, with a 16:8 h (light:dark) photoperiod, and 55 ± 5% relative humidity.…”

“…Laodelphax striatellus (Fallén) (small brown planthopper), Order: Hemiptera, Family: Delphacidae, is an important agricultural pest which attacks plants by sucking sap from pholem with its stylet. L. striatellus is also an important insect vector for many plant viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV) [1,2,3]. Among these viruses, RBSDV and RSV are known to cause devastating damages to multiple important cereal crops in many countries [3,4,5].…”