Molecular Characterization of Southern Rice Blacked‐Streaked Dwarf Virus (SRBSDV) from Vietnam

Southern rice black‐streaked dwarf virus (SRBSDV) is a novel putative member of the genus Fijivirus, family Reoviridae. We report here the genomic sequences of a Vietnamese isolate (SRBSDV‐V). The total genome of SRBSDV‐V has 29 115 nucleotides (nt), nine nt shorter than SRBSDV‐GD or ‐HN, but similar in organization to these two Chinese isolates. Nucleotide diversities among SRBSDV isolates were much lower than those among the corresponding ORFs of the available RBSDV isolates and there was a lower purifying selection pressure on SRBSDV than RBSDV, providing first molecular evidence for the view that SRBSDV is of recent origin. In studies of all available SRBSDV sequences, there was no obvious correlation between geographic distances and phylogenetic distribution. A high frequency of genetic recombination was found among both Chinese and Vietnam SRBSDV isolates, suggesting that recombination may play an important role in the molecular variation and evolution of SRBSDV.     

Specific and Rapid Detection of Lily symptomless virus and Arabis mosaic virus in Lily by Dual IC‐RT‐PCR

Lily symptomless virus (LSV) and Arabis mosaic virus (ArMV) cause severe losses of quantity and quality of lily flower and bulb production. Specificity, sensitivity and speed of detection methods for viruses need to be improved greatly to prevent LSV and ArMV from spreading from infected lilies. A dual IC‐RT‐PCR procedure for detection was developed in which the antibodies of LSV and ArMV were mixed and the mixture used to coat the PCR tubes. The particles of the two viruses were captured by the respective antibodies. Interference by other RNA viruses in infected lily was eliminated in the RT‐PCR. Also, an RNA extraction step was omitted. The dual IC‐RT‐PCR products of LSV and ArMV were 521 bp and 691 bp, respectively. The specificity of the method was validated; only LSV and ArMV of four viruses were detected by dual IC‐RT‐PCR. The sensitivity of the detection method is 1 mg leaf tissue and higher than DAS‐ELISA due to enrichment by dual immunocapture.     

Transmission by Laodelphax striatellus Fallen of Rice black‐streaked dwarf virus from Frozen Infected Rice Leaves to Healthy Plants of Rice and Maize

Rice black‐streaked dwarf virus (RBSDV) is transmitted naturally to important crops such as rice, maize, barley and wheat in a persistent manner by the planthoppers, Laodelphax striatellus, Unkanodes sapporona and Unkanodes albifascia. Insect vector transmission tests are the basis for identifying viral incidence, evaluating the resistance of varieties and selecting resistance sources for rice and maize breeding. A simple, rapid and reliable method is described by which virus‐free small brown planthoppers (L. striatellus) acquired RBSDV from frozen infected rice leaves and transmitted it to healthy rice and maize plants. After feeding on frozen infected rice leaves, the planthoppers were tested by RT‐PCR for the presence of virus after 10, 15, and 22 days, respectively. The percentages of RBSDV‐containing insects were 0, 25 and 71.43% of L. striatellus fed on frozen infected rice leaves compared to 0, 28.25 and 71.43% of L. striatellus fed on fresh infected rice leaves, respectively. In transmission tests, three of eight rice seedlings (37.5%) and four of eight maize seedlings (50%) were inoculated by the planthoppers that had fed previously on frozen leaves and had allowed a 22 days latent period and showed typical disease symptoms. As a positive control, four of eight rice seedlings (50%) and four of six maize seedlings (66.67%) became infected. All rice and maize plants expressing disease symptoms were identified as virus‐positive by RT‐PCR. These results indicated that the planthoppers acquired RBSDV from frozen infected leaves and transmitted the virus to healthy plants.     

Detection of Garlic Viruses Using SYBR Green Real‐time Reverse Transcription‐Polymerase Chain Reaction

SYBR Green real‐time RT‐PCR assay was developed and optimized for the sensitive detection of Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV), Shallot latent virus (SLV) and Mite‐borne filamentous virus (MbFV). The polyvalence of the designed primers was tested on 50 genotypes of garlic (Allium sativum L.) which originated from different countries. Plasmid standards were prepared and used as positive standards. The efficiencies of all reactions were 97, 93, 99, 98 and 87% for OYDV, LYSV, SLV, GCLV and MbFV standards, respectively. The detection limit for OYDV, LYSV and GCLV was as low as five gene copies, for SLV it was 15 gene copies and for MbFV it was 130 gene copies. In comparison with ELISA, more virus‐positive garlic accessions were detected with LYSV and GCLV by SYBR Green‐based real‐time RT‐PCR assay. This method was shown to be a more suitable tool for the detection of highly variable pathogens, such as garlic viruses.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

抗南方水稻黑条矮缩病水稻光温敏核不育系的筛选和鉴定

对东乡野生稻(Oryza rufipogon Griff.)3个生态群落株系及协青早B//协青早B/东乡野生稻的BC1F6株系进行了南方水稻黑条矮缩病抗性鉴定,筛选出抗性较好的种质资源。利用筛选到的协青早B//协青早B/东乡野生稻抗性株系,与光温敏核不育系C47S杂交转育,鉴定筛选到6份抗性较好的光温敏核不育系,为选育抗南方水稻黑条矮缩病的两系杂交稻组合奠定了材料基础;同时研究发现,来源于东乡野生稻的对南方水稻黑条矮缩病的抗性可能由数量性状基因控制。     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Development of reverse transcription loop‐mediated isothermal amplification assay for sensitive and rapid detection of Hosta virus X

The one‐step real‐time turbidity loop‐mediated isothermal amplification assay (RealAmp) was developed to detect Hosta virus X (HVX), the most devastating threat to hosta industry. The reaction was performed in a single tube at 63°C for 15 min, and real‐time turbidimetry was used to monitor the amplification results. Specificity and sensitivity analyses demonstrated that this RealAmp method was sensitive as real‐time TaqMan RT‐PCR and about 100‐fold higher than conventional RT‐PCR with no cross‐reaction with other viral pathogens. Field samples detection showed that HVX could be identified effectively with this method. Overall, this RealAmp assay for HVX detection was simple, specific, sensitive, convenient and time‐saving and could assist in the quarantine measures for prevention and control of the disease caused by HVX.     

Efficient Inoculation of Rice black‐streaked dwarf virus to Maize Using Laodelphax striatellus Fallen

Maize rough dwarf disease caused by Rice black‐streaked dwarf virus (RBSDV) is the most important disease of maize in China. Although deploying disease resistant hybrids would be the most effective way to control the disease, development of resistant hybrids has been limited by virus transmission rates that are too low for effective screening. An efficient inoculation technique for RBSDV was developed using Laodelphax striatellus Fallen, in which a virus‐free planthopper colony was developed and viruliferous planthoppers were obtained by allowing a 3‐ to 4‐day acquisition access period on RBSDV‐infected wheat plants. Planthoppers were then allowed a 25‐ to 28‐day latent period on wheat seedlings followed by a 3‐day inoculation access period on two‐to‐three‐leaf stage maize seedlings. By 35 days postinoculation, susceptible hybrid ‘Zhengdan 958’, inbred lines of ‘Ye 107’ and ‘Ye 478’ plants showed 100% RBSDV infection with symptoms of stunting plants, darkening leaves and white waxy swellings on underside of leaves. At tasseling stage, average disease indices were from 96.4 to 100.0%. Enzyme‐linked immunosorbent assays were correlated with the presence of symptoms. The high efficiency of RBSDV transmission obtained using this technique provides a reliable procedure to screen for RBSDV resistance in maize.     

Development and preliminary application of a triplex real‐time polymerase chain reaction assay for evaluating predation on three planthoppers in a rice ecosystem

A multiplex real‐time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white‐backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real‐time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white‐backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross‐reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real‐time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.     

Detection of Helicobacter pylori in stool samples of young children using real‐time polymerase chain reaction

Background

The aims of this study were to develop and validate a multiplex real‐time polymerase chain reaction (q‐PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori‐positive samples.

Materials and methods

Archived stool samples from 188 children aged 6‐9 years and 272 samples of 92 infants aged 2‐18 months were tested for H. pylori antigens using enzyme immunoassay (EIA). A multiplex q‐PCR assay was designed to detect H. pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q‐PCR and EIA.

Results

Laboratory validation of the q‐PCR assay using quantitated H. pylori ATCC 43504 extracted DNA showed S‐shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross‐reactivity with other bacterial pathogens was noted. Applying the multiplex q‐PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H. pylori infection was 50% (95% CI 43%‐57%) by q‐PCR (urease cycle threshold <44) vs 59% (95% CI 52%‐66%) by EIA. Kappa coefficient was .80 (P < .001) and .44 (P < .001) for children aged 6‐9 years and 2‐18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing.

Conclusions

The developed q‐PCR can be used as a cotechnique to enhance the accuracy of H. pylori detection in epidemiological studies and in clinical settings.