Using non-radioactive probes on plants: a few examples

Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a pest in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes. © 1998 John Wiley & Sons, Ltd.     

A rapid detection of tomato yellow leaf curl virus using recombinase polymerase amplification-lateral flow dipstick assay

Tomato yellow leaf curl disease which is caused by Tomato yellow leaf curl virus (TYLCV) is economically important and a widely spread tomato disease in China. Rapid and accurate detection methods are important in the control TYLCV. Here, a rapid method was developed to identify TYLCV on the basis of recombinase polymerase amplification (RPA) that can be visualized in 5 min using lateral flow dipsticks. The sensitivity and the specificity of this method were evaluated. This method can detect 0·5 pg DNA after 30 min at 37°C without any expensive instrumentation. In addition, it showed higher sensitivity than a PCR method when purified DNA was used. Moreover, the TYLCV was specifically detected, whereas other viruses infecting tomato produced negative results. The crude tomato extracts used in this assay has potential application in minimally equipped plant clinic laboratories. This method will facilitate the early and rapid detection of TYLCV for the timely application of control measures.     

Detection of sub-picogram quantities of specific DNA sequences on blot hybridization with biotinylated probes.

A sensitive method for detecting biotinylated DNA probes on dot and Southern blots is described which is based on the principle outlined by Leary et al (1). This system has two main components: detection of biotinylated DNA by a two-step procedure with streptavidin and poly(alkaline phosphatase); and blocking background with Tween 20. 32fg and 80fg of lambda phage DNA was detected on dot and Southern blot hybridizations respectively. 150fg of beta-globin was detected on Southern blots of genomic DNA. This method is fast, reproducible and can detect single copy genes in 0.25 micrograms genomic DNA on Southern blots.     

The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.     

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.Abbreviations BA benzyladenine - CMV cucumber mosaic virus - NAA naphthaleneacetic acid - TMV tobacco mosaic virus - TYLCV tomato yellow leaf curl virus     

A rapid PCR method to discriminate between Tomato yellow leaf curl virus isolates

Tomato yellow leaf curl virus (TYLCV) was recently divided into two different species: Tomato yellow leaf curl virus‐Israel (TYLCV‐Is) and Tomato yellow leaf curl virus‐Sardinia (TYLCV‐Sar). There are no rapid methods by which TYLCV viruses may be assigned to either TYLCV‐Is or TYLCV‐Sar species. In the present work, using an extensive alignment of begomovirus sequences, TYLCV‐specific primers were designed and tested which allow the specific amplification of DNA fragments from any isolate of TYLCV. Also, a primer was designed and tested which allows the specific amplification of TYLCV‐Sar. Furthermore, a combination of these primers was selected to develop a duplex PCR method, which has the potential to detect either TYLCV‐Is or TYLCV‐Sar. The PCR methods were also highly effective with minimal sample preparation and allowed direct amplification of TYLCV from infected leaf extracts. This approach may be used in the laboratory as a tool for rapid, large‐scale diagnostics of TYLCV‐infected samples.     

Reservoir Weed Hosts of Tomato Yellow Leaf Curl Begomovirus from Tanzania

A survey was initiated to detect tomato yellow leaf curl virus (TYLCV) and identify its reservoir weed hosts in six regions (Arusha, Morogoro, Dodoma, Iringa, Kilimanjaro and Dar es Salaam) in Tanzania. Three farms were randomly selected in each region. Assessment of TYLCV incidence was done by relating the number of infected tomato plants to the total number of plants assessed along a diagonal in five quadrants measuring 4m ‐ 4m in size (one at each corner of the farm and one at the centre). Disease severity was scored on a scale of 0 to 4 (where 0 = no symptoms and 4 = very severe symptoms). Within and outside each farm, weeds showing TYLCV-like symptoms were collected and either squash-blotted, dot-blotted or both on nylon membranes. The membranes were hybridized with DIG-labelled probe synthesized for the detection of TYLCV from Sardinia (TYLCV-Sar) following standard protocols. Selected plant species were experimentally inoculated with screenhouse cultures of TYLCV representative isolates from the six regions using Bemisia tabaci to determine their host status. Results indicated that TYLCV incidence and severity were significantly higher (P = 0.05) in Dodoma region than the rest of the regions. In Iringa region, the incidence and severity of TYLCV were the lowest of all regions. TYLCV was detected in 12 of the 17 dot-blotted samples and in all the 21 squashed samples using the non-radioactively labelled riboprobes. Similarly, five plant species (Capsicum annuum, Datura stramonium, Lycopersicon esculentum, Nicotiana glutionsa and N. tabacum) tested in the screenhouse were infected by the six TYLCV isolates used. It is recommended that weeds within and outside tomato farms be removed to eliminate or reduce sources of virus inoculum. The dot and squash blot techniques are convenient for field detection of the virus, and are especially useful for the detection of early and latent infections so that management strategies can be initiated and implemented.     

Enhanced chemiluminescent method for the detection of DNA dot-hybridization assays

A simple enhanced chemiluminescent procedure for the quantitation of DNA hybridization to dot blots is described. The method utilizes DNA probes labeled with biotin, which are detected using a biotinylated streptavidin-horseradish peroxidase complex. The peroxidase enzyme then takes part in an enhanced chemiluminescent reaction with luminol, peroxide, and an enhancer. The method can be used to give quantitative results using a photomultiplier tube or qualitative results by recording the light emission on instant photographic film.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

Screening for the presence of the insertion sequence IS1 in the genus Bacteroides

Abstract A specific DNA probe, containing a conserved region of the insertion sequence IS1, was hybridised to dot blots of total genomic DNA from 2 oral and 5 intestinal Bacteroides spp. Using Escherichia coli K12 as a positive control and Pseudomonas aeruginosa as a negative control, DNA homologous to the probe could not be detected in Bacteroides corporis, Bacteroides intermedius, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron or 2 strains of Bacteroides fragilis . The total DNA included plasmid DNA of 30.2, 42.7 and 42.7 MDa from B. fragilis, B. intermedius and B. corporis , respectively.
IS1 is commonly found in members of the Enterobacteriaceae, and it was concluded that the 2 groups of bacteria are not closely related.     

Detection of a common BOLA-DRB3 deletion by sequence-specific oligonucleotide typing

The bovine MHC class II BoLA-DRB3*2A has an amino acid deletion of unknown function at codon 65 in the second exon, which codes for the antigen-binding site. Sequence-specific oligonucleotides were designed based on published nucleotide sequences on BoLA-DRB3 alleles, and used to detect this deletion in 51 Hereford cattle. Probes 65+ and 65- detect the presence or absence of codon 65 respectively. Oligonucleotide probes were labelled with Digoxigenin (DIG), hybridized to dot blots of BOLA-DRB3 exon 2 polymerase chain reaction (PCR) product, and detected by chemiluminescence. Of the 51 animals screened, two were homozygous and 11 were heterozygous for the deletion at codon 65. The methodology described here provides the necessary tools to screen rapidly for this deletion in a large number of animals in order to study its effect on antigen binding and immune response.     

聚合酶链式反应结合Southern杂交检测外源DNA转化根癌农杆菌

本文介绍检测外源ＤＮＡ转化根癌农杆菌的新方法,将ＰＣＲ结合Ｓ杂交进行检测,与直接进行菌落杂交和点杂交相比,更为灵敏省时。     

Differential responses between a vector species Bemisia tabaci and a nonvector species Trialeurodes vaporariorum following ingestion of tomato yellow leaf curl virus

In transmitting plant viruses, insect vectors undergo physiological and behavioral alterations. The whitefly Bemisia tabaci is a vector of tomato yellow leaf curl virus (TYLCV), causing severe damages to various horticultural crop plants. To determine whether whitefly alteration is specific to vector species, the responses to TYLCV ingestion were compared between B. tabaci and Trialeurodes vaporariorum, a nonvector for TYLCV. The two species were reared on TYLCV‐infected and noninfected tomato, a host of TYLCV, and their longevity and fecundity were determined while rearing in either tomato or eggplant, a nonhost of TYLCV. TYLCV‐ingested B. tabaci increased their developmental rates but reduced fecundity when they were reared in either tomato or eggplant compared with those of TYLCV‐free ones. In contrast, TYLCV‐ingested T. vaporariorum did not show any of the aforementioned changes when reared on both plant species. In addition, TYLCV‐ingested B. tabaci increased their levels of three heat shock protein genes ( hsp20, hsp70, and hsp90) against thermal stress, whereas TYLCV‐ingested T. vaporariorum did not. The presence of TYLCV virions was identified in two colonies of both species via polymerase chain reaction analysis. TYLCV was detected in the whole body, saliva, and eggs of B. tabaci, while TYLCV was detected only in the whole body but not in the saliva and eggs of T. vaporariorum. The present results strongly indicated that TYLCV specifically manipulate physiological processes of the vector species, B. tabaci.     

Sequential chemiluminescent detection of target DNAs without stripping and reprobing.

We present a simple method for sequential chemiluminescent detections of two different DNA loci on a single Southern blot. First, an enzyme-linked DNA probe for a unique sequence is detected with a horse-radish peroxidase (HRP) substrate followed by the detection of another enzyme-linked DNA probe for a different unique sequence with an alkaline phosphatase (AP) substrate that simultaneously inhibits the chemiluminescence generated by HRP. Such sequential detection steps eliminate the need to strip and reprobe blots and can be performed with no intervening steps.     

Rapid spread of tomato yellow leaf curl virus in China is aided differentially by two invasive whiteflies

Background

Tomato yellow leaf curl virus (TYLCV) was introduced into China in 2006, approximately 10 years after the introduction of an invasive whitefly, Bemisia tabaci (Genn.) B biotype. Even so the distribution and prevalence of TYLCV remained limited, and the economic damage was minimal. Following the introduction of Q biotype into China in 2003, the prevalence and spread of TYLCV started to accelerate. This has lead to the hypothesis that the two biotypes might not be equally competent vectors of TYLCV.

Methodology/Principal Findings

The infection frequency of TYLCV in the field-collected B. tabaci populations was investigated, the acquisition and transmission capability of TYLCV by B and Q biotypes were compared under the laboratory conditions. Analysis of B. tabaci populations from 55 field sites revealed the existence of 12 B and 43 Q biotypes across 18 provinces in China. The acquisition and transmission experiments showed that both B and Q biotypes can acquire and transmit the virus, however, Q biotype demonstrated superior acquisition and transmission capability than its B counterparts. Specifically, Q biotype acquired significantly more viral DNA than the B biotype, and reached the maximum viral load in a substantially shorter period of time. Although TYLCV was shown to be transmitted horizontally by both biotypes, Q biotype exhibited significantly higher viral transmission frequency than B biotype. Vertical transmission result, on the other hand, indicated that TYLCV DNA can be detected in eggs and nymphs, but not in pupae and adults of the first generation progeny.

Conclusions/Significance

These combined results suggested that the epidemiology of TYLCV was aided differentially by the two invasive whiteflies (B and Q biotypes) through horizontal but not vertical transmission of the virus. This is consistent with the concomitant eruption of TYLCV in tomato fields following the recent rapid invasion of Q biotype whitefly in China.     

Chinese tomato yellow leaf curl virus--a new species of geminivirus

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Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes

Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions.     

Low frequency of horizontal and vertical transmission of two begomoviruses through whiteflies exhibits little relevance to the vector infectivity

Transmissions of plant viruses between individuals of their vector insects through mating are rare events. Recently, three begomoviruses were found to be transmitted between males and females of the whitefly Bemisia tabaci through mating, and two viruses were shown to be transmitted transovarially to progeny. However, results between reports were not consistent. Here we examined the horizontal and vertical transmission of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl China virus (TYLCCNV) by the B and Q biotypes of B. tabaci, using virus isolates and whitefly colonies established recently in China. Both TYLCV DNA and TYLCCNV DNA were shown to be transmitted horizontally and vertically by each of the two biotypes of the whitefly, but frequency of transmission was usually low. In transovarial transmission, virus DNA was detected in eggs and nymphs but not in the adults of the first generation progeny, except in the combination of TYLCV and Q biotype whitefly where 2–3% of the offspring adults contained the virus DNA. We also showed that the first generation adults, which developed from eggs of viruliferous whiteflies, were not infective to plants. These results demonstrated that for the viruses and whiteflies tested here low frequency of horizontal and vertical transmission can be expected but these two modes of transmission are unlikely to have much epidemiological relevance in the field.     

Imaging of chemiluminescent signals with cooled CCD camera systems

We investigated imaging of chemiluminescent signals from 1,2-dioxetanes with cooled CCD cameras. Non-radioactive detection methods for biomolecules utilizing these chemiluminescent substrates for alkaline phosphatase have been developed. Applications which have been successfully adapted to this technology include Southern and Northern blotting, immunoblotting, ELISA methods and DNA sequencing. Dephosphorylation of the dioxetane CSPD by alkaline phosphatase generates an unstable anion that decomposes resulting in light production. The wavelength of the emitted light is approximately 460nm. We have utilized Photometrics Star and MXC 200L cooled CCD cameras for direct imaging of chemiluminescent signals. Benefits of utilizing a CCD detector include rapid data digitization and more accurate quantitation of chemiluminescent signals compred to film-based densitometry owing to the significantly greater dynamic range. Chemiluminescent images from dot blots of biotinylated DNA, Southern blots and DNA sequencing gel blots were obtained. In a chemiluminescent microtitre plate assay, serial dilutions of alkaline phosphatase spanning four orders of magnitude can be detected. Our results indicate that the digitization of chemiluminescent signal data with cooled CCD cameras is an excellent alternative to ³²P detection methods utilizing storage phosphor screen imaging systems.     

Development and Evaluation of Simple Dot–Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food

The present study was aimed to develop and evaluate dot–blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot–blot format, recombinant SEA was directly coated on NCM dot–blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot–blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot–blot assays exhibited a sensitivity of ~48 ng ml^?1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot–blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot–blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.