Dataset Information

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

ABSTRACT: The 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.We 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.A 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.

SUBMITTER: Xu Q

PROVIDER: S-EPMC5415734 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Xu Qiufang Q Liu Haoqiu H Yuan Pingping P Zhang Xiaoxia X Chen Qingqing Q Jiang Xuanli X Zhou Yijun Y

Virology journal 20170503 1

<h4>Background</h4>The 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 trad ...[more]

PMID: 28468626

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Project description:The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus. The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 (LsRPL5) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin (Ls?-TUB) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus. LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus, while beta-tubulin (Ls?-TUB) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1-silenced L. striatellus. The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus. The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus.

Project description:BACKGROUND: The small brown planthopper (Laodelphax striatellus) is an important agricultural pest that not only damages rice plants by sap-sucking, but also acts as a vector that transmits rice stripe virus (RSV), which can cause even more serious yield loss. Despite being a model organism for studying entomology, population biology, plant protection, molecular interactions among plants, viruses and insects, only a few genomic sequences are available for this species. To investigate its transcriptome and determine the differences between viruliferous and naïve L. striatellus, we employed 454-FLX high-throughput pyrosequencing to generate EST databases of this insect. RESULTS: We obtained 201,281 and 218,681 high-quality reads from viruliferous and naïve L. striatellus, respectively, with an average read length as 230 bp. These reads were assembled into contigs and two EST databases were generated. When all reads were combined, 16,885 contigs and 24,607 singletons (a total of 41,492 unigenes) were obtained, which represents a transcriptome of the insect. BlastX search against the NCBI-NR database revealed that only 6,873 (16.6%) of these unigenes have significant matches. Comparison of the distribution of GO classification among viruliferous, naïve, and combined EST databases indicated that these libraries are broadly representative of the L. striatellus transcriptomes. Functionally diverse transcripts from RSV, endosymbiotic bacteria Wolbachia and yeast-like symbiotes were identified, which reflects the possible lifestyles of these microbial symbionts that live in the cells of the host insect. Comparative genomic analysis revealed that L. striatellus encodes similar innate immunity regulatory systems as other insects, such as RNA interference, JAK/STAT and partial Imd cascades, which might be involved in defense against viral infection. In addition, we determined the differences in gene expression between vector and naïve samples, which generated a list of candidate genes that are potentially involved in the symbiosis of L. striatellus and RSV. CONCLUSIONS: To our knowledge, the present study is the first description of a genomic project for L. striatellus. The identification of transcripts from RSV, Wolbachia, yeast-like symbiotes and genes abundantly expressed in viruliferous insect, provided a starting-point for investigating the molecular basis of symbiosis among these organisms.

Dataset Information

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

Publications

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

Similar Datasets

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