Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.     

Species‐specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

Inteins (internal proteins) are self‐splicing transportable genetic elements present in conserved regions of housekeeping genes. The study highlights the importance of intein as a potential diagnostic marker for species‐specific identification of Candida tropicalis, a rapidly emerging opportunistic human pathogen. Initial steps of primer validation, sequence alignment, phylogenetic tree analysis, gel electrophoresis and real‐time polymerase chain reaction (PCR) assays were performed to confirm the specificity of the designed primers. The primers were selective for C. tropicalis with 100% inclusivity and showed no cross‐species or cross‐genera matches. The established technique is a prototype for developing multifaceted PCR assays and for point‐of‐care testing in near future.

Significance and Impact of the Study

Development of molecular markers for specific detection of microbial pathogens using real‐time polymerase chain reaction (PCR) is an appealing and challenging technique. A real‐time PCR is an emerging technology frequently used to detect the aetiologic agents. In recent times, designing species‐specific primers for pathogen detection is gaining momentum. The method offers rapid, accurate and cost‐effective strategy to identify the target, thus providing sufficient time to instigate appropriate chemotherapy. The study highlights the use of intein DNA sequence as molecular markers for species‐specific identification of Candida tropicalis. The study also offers a prototype model for developing multifaceted PCR assays using intein DNA sequences, and provides a developmental starting point for point‐of‐care testing in near future.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

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.     

Detection of Puccinia pelargonii‐zonalis‐infected Geranium Tissues and Urediniospores

The occurrence of geranium rust (caused by Puccinia pelargonii‐zonalis) in commercial greenhouses can result in unmarketable plants and significant economic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii‐zonalis‐infected tissues or urediniospores on greenhouse‐grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii‐zonalis. The primers amplified a 131‐bp product from genomic DNA from each isolate of P. pelargonii‐zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii‐zonalis DNA in conventional PCR and at 1 pg/ml using real‐time PCR. The detection threshold was 10² urediniospores/ml for real‐time PCR and 10⁴ urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii‐zonalis DNA was amplified by real‐time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non‐inoculated leaves. Conventional and real‐time PCR did not detect P. pelargonii‐zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conventional and real‐time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subsequent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses.     

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.     

Multiplex real‐time PCR assays for detection of four seedborne spinach pathogens

Aims

To develop multiplex TaqMan real‐time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach.

Methods and Results

Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real‐time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real‐time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed.

Conclusions

The real‐time PCR assays were species‐specific and sensitive. Singular or multiplex real‐time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed.

Significance and Impact of the Study

The freeze‐blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real‐time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.     

Preparation of aminated core‐shell fluorescent nanoparticles and their application to the synchronous fluorescence determination of γ‐globulin

Amino‐modified silica nanoparticles (FSNPs) doped with fluorescein isothiocyanate (FITC) were synthesized by using an aqueous core of reverse‐micelle microemulsion as the nanoreactor in an easy one‐pot method. Due to the FITC conjugating with (3‐aminopropyl)triethoxysilane (APTS), the nanoparticles prevent the FITC from leaching from the silica matrix when immersed in aqueous solution. SEM, FTIR, fluorescence lifetime, a photobleaching experiment and synchronous fluorescence spectra were used to characterize the FSNPs. The synchronous fluorescence signal of FSNPs was enhanced when trace amounts of γ‐globulin (γ‐G) were added. Under the optimal experimental conditions, the enhanced fluorescence intensity (ΔF) was linear with the concentration of γ‐G (c) in the range 0.3–4.8 µg/mL, with a detection limit of 0.04 µg/mL. The proposed method is simple, sensitive for the determination of trace amounts of γ‐G and used to determine the content of γ‐G in synthetic samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

Performance comparison of genetic markers for high‐throughput sequencing‐based biodiversity assessment in complex communities

Metabarcode surveys of DNA extracted from environmental samples are increasingly popular for biodiversity assessment in natural communities. Such surveys rely heavily on robust genetic markers. Therefore, analysis of PCR efficiency and subsequent biodiversity estimation for different types of genetic markers and their corresponding primers is important. Here, we test the PCR efficiency and biodiversity recovery potential of three commonly used genetic markers – nuclear small subunit ribosomal DNA (18S), mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (mt16S) – using 454 pyrosequencing of a zooplankton community collected from Hamilton Harbour, Ontario. We found that biodiversity detection power and PCR efficiency varied widely among these markers. All tested primers for COI failed to provide high‐quality PCR products for pyrosequencing, but newly designed primers for 18S and 16S passed all tests. Furthermore, multiple analyses based on large‐scale pyrosequencing (i.e. 1/2 PicoTiter plate for each marker) showed that primers for 18S recover more (38 orders) groups than 16S (10 orders) across all taxa, and four vs. two orders and nine vs. six families for Crustacea. Our results showed that 18S, using newly designed primers, is an efficient and powerful tool for profiling biodiversity in largely unexplored communities, especially when amplification difficulties exist for mitochondrial markers such as COI. Universal primers for higher resolution markers such as COI are still needed to address the possible low resolution of 18S for species‐level identification.     

SP‐Designer: a user‐friendly program for designing species‐specific primer pairs from DNA sequence alignments

SP‐Designer is an open‐source program providing a user‐friendly tool for the design of specific PCR primer pairs from a DNA sequence alignment containing sequences from various taxa. SP‐Designer selects PCR primer pairs for the amplification of DNA from a target species on the basis of several criteria: (i) primer specificity, as assessed by interspecific sequence polymorphism in the annealing regions, (ii) the biochemical characteristics of the primers and (iii) the intended PCR conditions. SP‐Designer generates tables, detailing the primer pair and PCR characteristics, and a FASTA file locating the primer sequences in the original sequence alignment. SP‐Designer is Windows‐compatible and freely available from http://www2.sophia.inra.fr/urih/sophia_mart/sp_designer/info_sp_designer.php .     

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop‐mediated isothermal amplification

Aim: The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. Methods: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real‐time polymerase chain reaction (FQ‐PCR). Results: The results were as follows. (1) Serovar‐specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20 min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl^?1; thus, the sensitivity and specificity of this assay is similar to those of the FQ‐PCR. Conclusions: LAMP is a high‐throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. Significance and impact of the study: This is the first study involving the use of LAMP to detect Salmonella serovar‐specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.     

Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

Aims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real‐time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP‐154 and CFN‐535 in the rhizosphere. Methods and Results: Primers were designed based on strain‐specific SCAR markers and were screened for successful amplification of target strain and absence of cross‐reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real‐time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 × 10³ (for UAP‐154) and 4 × 10⁴ CFU g^?1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10⁴ to 10⁸ CFU g^?1 soil. Conclusion: BOX‐based SCAR markers were useful to find primers for strain‐specific real‐time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation‐independent monitoring methods were lacking. The real‐time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP‐154 and CFN‐535.     

Arbovirus and insect‐specific virus discovery in Kenya by novel six genera multiplex high‐resolution melting analysis

A broad diversity of arthropod‐borne viruses (arboviruses) of global health concern are endemic to East Africa, yet most surveillance efforts are limited to just a few key viral pathogens. Additionally, estimates of arbovirus diversity in the tropics are likely to be underestimated as their discovery has lagged significantly over past decades due to limitations in fast and sensitive arbovirus identification methods. Here, we developed a nearly pan‐arbovirus detection assay that uses high‐resolution melting (HRM) analysis of RT–PCR products from highly multiplexed assays to differentiate broad diversities of arboviruses. We differentiated 15 viral culture controls and seven additional synthetic viral DNA sequence controls, within Flavivirus, Alphavirus, Nairovirus, Phlebovirus, Orthobunyavirus and Thogotovirus genera. Among Bunyamwera, sindbis, dengue and Thogoto virus serial dilutions, detection by multiplex RT–PCR‐HRM was comparable to the gold standard Vero cell plaque assays. We applied our low‐cost method for enhanced broad‐range pathogen surveillance from mosquito samples collected in Kenya and identified diverse insect‐specific viruses, including a new clade in anopheline mosquitoes, and Wesselsbron virus, an arbovirus that can cause viral haemorrhagic fever in humans and has not previously been isolated in Kenya, in Culex spp. and Anopheles coustani mosquitoes. Our findings demonstrate how multiplex RT–PCR‐HRM can identify novel viral diversities and potential disease threats that may not be included in pathogen detection panels of routine surveillance efforts. This approach can be adapted to other pathogens to enhance disease surveillance and pathogen discovery efforts, as well as the study of pathogen diversity and viral evolutionary ecology.     

Development of Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction primers based on the nucleotide sequence of rpoB

In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.     

Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P. nicotianae is essential for controlling these diseases. In this study, primers based on the Ras‐related protein gene (Ypt1) of P. nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P. nicotianae isolates, 45 isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross‐reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100 fg and 10 fg genomic DNA per 25‐μl reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P. nicotianae‐infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P. nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P. nicotianae LAMP visual detection.     

Loop‐mediated isothermal amplification assay targeting the blaCTX‐M9 gene for detection of extended spectrum β‐lactamase‐producing Escherichia coli and Klebsiella pneumoniae

Extended‐spectrum β‐lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β‐lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL‐producing bacteria is necessary for identification, prevention and treatment. Loop‐mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL‐producing bacteria by a LAMP technique. ESBLs‐producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double‐disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla_CTX‐M9 gene was designed based on bla_CTX‐M9 from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder‐like patterns of band sizes from 226 bp of the bla_CTX‐M9 DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla_CTX‐M9 was greater than that of the PCR method by at least 10,000‐fold. These results showed that the LAMP primers specifically amplified only the bla_CTX‐M9 gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.