A real‐time PCR assay for improved rapid,specific detection of Cryphonectria parasitica

Cryphonectria parasitica, an ascomycete fungus, is the causal agent of chestnut blight. This highly destructive disease of chestnut trees causes significant losses, and is therefore a regulated pathogen in Europe. Existing methods for the detection of C. parasitica include morphological identification following culturing, or PCR; however, these are time‐consuming resulting in delays to diagnosis. To allow improved detection, a new specific real‐time PCR assay was designed to detect C. parasitica directly from plant material and fungal cultures, and was validated according to the European Plant Protection Organisation (EPPO) standard PM 7/98. The analytical specificity of the assay was tested extensively using a panel of species taxonomically closely related to Cryphonectria, fungal species associated with the hosts and healthy plant material. The assay was found to be specific to C. parasitica, whilst the analytical sensitivity of the assay was established as 2 pg µL^?1 of DNA. Comparative testing of 63 samples of naturally infected plant material by the newly developed assay and traditional morphological diagnosis demonstrated an increased diagnostic sensitivity when using the real‐time PCR assay. Furthermore the assay is able to detect both virulent and hypovirulent strains of C. parasitica. Therefore the new real‐time PCR assay can be used to provide reliable, rapid, specific detection of C. parasitica to prevent the accidental movement of the disease and to monitor its spread.     

Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors

Three real‐time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr‐V, 16Sr‐X and 16Sr‐XII have been developed. Ribosomal‐based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real‐time PCR. For group‐specific detection of FD phytoplasma, ribosomal‐based primers fAY/rEY, specific for 16Sr‐V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non‐ribosomal and nitroreductase DNA sequences, respectively. SYBR^® Green I detection coupled with melting curve analysis was used in each group‐specific protocol. Field‐collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real‐time PCR assays. The diagnostic efficiency of each group‐specific protocol was compared with well‐established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real‐time PCR from DNAs of European aster yellows (16Sr‐I), elm yellows (16Sr‐V), stolbur (16Sr‐XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group‐specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real‐time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group‐specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.     

Quantification of Fusarium culmorum in Wheat and Barley Tissues Using Real-Time PCR in Comparison with DON Content

A real‐time polymerase chain reaction (PCR) assay was developed for the specific detection of Fusarium culmorum in infected seeds. Primers and TaqMan minor groove binder probe were derived from the sequences of a F. culmorum specific PCR product. The specificity of the assay was confirmed by test in seven Fusarium species and 21 non‐Fusarium fungal species. With serial dilutions of purified genomic DNA from F. culmorum isolate B as the template, the detection limit of the assay was found to be 0.9 pg of fungal genomic DNA per reaction. A significant correlation ( = 0.982) and collinearity was found between DNA concentration and Ct (cycle threshold) values of real‐time PCR assay with serial diluted DNAs extracted from three seed samples with different deoxynivalenol (DON) content. Eight barley and nine wheat varieties infected by F. culmorum isolate B were evaluated in 1 (barley samples) and in 4 years (wheat samples). The results of real‐time PCR analysis and enzyme‐linked immunosorbent assay testing for DON content were compared and a significant correlation was found for barley samples (r² = 0.935). Concerning wheat we found rather complicated relationship between Ct values and DON contents influenced by environmental conditions of field trials. The real‐time PCR assay was found to be highly specific and sensitive. It could be used in phytopathological studies and praxis.     

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

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.     

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.     

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.     

Evaluation of the efficacy of immunomagnetic separation for the detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds

Aims: To evaluate the effectiveness of the optimized immunomagnetic separation (IMS)‐plating protocol in relation to other culture, serological and molecular techniques currently used for Clavibacter michiganensis subsp. michiganensis in seed‐testing laboratories. Methods and results: Bacterial suspensions, tomato seed extracts spiked with the pathogen and naturally infected seeds were IMS‐plated for the detection of C. m. subsp. michiganensis. These results were compared with plating on general (YPGA) and semiselective (mSCM) media, double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA), immunofluorescent assay (IF) or polymerase chain reaction (PCR). Different seed lots and pathogen strains were also tested. IMS‐plating allowed the detection of less than 10 CFU ml^?1 of pathogen in all assayed samples. The mSCM medium provided positive results for 10 CFU ml^?1 in naturally infected seeds, but up to 14 days was necessary for the typical colonies of the target to be come visible. By serological techniques, 10³ and up to 10⁴ CFU ml^?1 were detected by IF and ELISA, respectively. DNA extraction was required to obtain positive results by PCR in seed extracts containing 10³ CFU ml^?1 or more. Conclusions: Among the evaluated methods, IMS‐plating provided the best results regarding sensitivity and specificity for C. m. subsp. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. Significance and impact of the study: IMS‐plating increases isolation rates of C. m. subsp. michiganensis and could improve standard protocols currently used for routine analysis.     

Triplex real‐time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT‐PCR method was established. This triplex real‐time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real‐time PCR across a broad dynamic range of 10²–10⁷ copies/assay using plasmid DNA standards. The limit of detection was 10² copies/assay. The quantitative value was comparable with that of monoplex real‐time PCR of stool samples. Our triplex real‐time PCR is useful for detection of NoV and SaV infections.     

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.     

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.     

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

Detection of Latent Infection of Wheat Leaves Caused by Blumeria graminis f.sp. tritici Using Nested PCR

Wheat powdery mildew caused by Blumeria graminis f.sp. tritici (Bgt) is an important and destructive disease worldwide. Detection of latent infection of wheat seedlings is critical to estimate initial inoculum potential of epidemics in the fields. To improve the conventional method, a nested PCR approach had been established in this study to detect latent infections of wheat leaves caused by Bgt. The DNA primer sets including external and internal primer pairs for the nested PCR were designed followed by testing their specificities to Bgt by using Bgt and other fungal species of wheat. Sensitivity test demonstrated that the nested PCR could detect as low as 0.1 fg template DNA and about 10,000 times more sensitive than the standard PCR. Results of artificial inoculation experiments showed that the nested PCR assay can detect a low level of latent infection of wheat seedlings 2 days earlier than did standard PCR. The incidences of latent infection of wheat seedlings determined by the nested PCR linearly correlated with those by the conventional incubation method (r² = 0.66, P = 0.0023). The incidences of latent infection detected with nested PCR were higher than that with the conventional method. This study provides an accurate method to efficiently estimate the initial inoculum potential of wheat powdery mildew epidemics in the fields.     

Quantification of Campylobacter spp. in chicken carcass rinse by real‐time PCR

Aims: In this study, a real‐time quantitative polymerase chain reaction (PCR) method was examined for its ability to quantify Campylobacter spp. in chicken carcass rinses and compared with bacteriological culturing. Methods and Results: The linearity of the real‐time PCR quantification protocol was assessed on pure DNA. The amplification efficiency was 100% and the square regression coefficient (R²) was 0·998. Quantification was linear over at least 7 log units. Using a crude cell lysate gave the highest sensitivity and the detection limit of the method was 3·3 log CFU per carcass. The statistical correlation between the bacteriological enumeration and the real‐time quantitative (Q)‐PCR determined using chicken carcasses sampled at the end of the slaughter line was 0·733. The difference in detection levels was probably because of the detection of stressed, dead or viable but not culturable cells by Q‐PCR. Conclusion: The real‐time Q‐PCR method described in this study is a powerful tool for determining the number of Campylobacter cells on carcasses. Significance and Impact of the Study: The real‐time Q‐PCR method is available to quantify the Campylobacter contamination at the slaughterhouse level and could be used to evaluate primary production.     

Development and Validation of Conventional and Quantitative Polymerase Chain Reaction Assays for the Detection of Storage Rot Potato Pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata

The diseases pink rot, watery wound rot and gangrene are important storage rot diseases of potato associated predominantly with Phytophthora erythroseptica (P.), Pythium ultimum (Py.) and Phoma exigua (Phoma) var. foveata respectively. Reliable molecular‐based diagnostic tests are required that will not only allow unequivocal identification of symptoms but will further advance epidemiological studies of these potato diseases to increase our understanding and contribute to more effective management and control strategies to the potato industry. Primers and probes were designed in specific regions of the internal transcribed spacer (ITS) regions to develop conventional and real‐time quantitative polymerase chain reaction (PCR) assays able to detect all possible fungal and oomycete pathogens causing pink rot, watery wound rot and gangrene. The specificity of each diagnostic assay was rigorously tested with over 500 fungal/oomycete plant pathogen isolates from potato and reference culture collections, and both conventional and real‐time PCR methods produced similar results. In terms of sensitivity, the detection limits for real‐time PCR went below ag DNA levels compared with pg DNA levels with conventional PCR. The real‐time PCR assays developed to detect Phoma foveata and Py. ultimum on tubers were suitable for the comparative C_t method (ΔΔCt) of quantification using the cytochrome oxidase gene of potato as a normalizer assay; an advantage as the need for a standard curve is eliminated. Each assay detected Phoma species (var. foveata or exigua) from naturally infected tubers showing symptoms of gangrene, and P. erythroseptica or Py. ultimum were also detected following inoculation of Russet Burbank tubers. Each diagnostic assay developed could reliably detect and distinguish between the pink rot, watery wound rot and gangrene‐causing potato pathogens.     

Beneficial effect of GB virus C co-infection in Human Immunodeficiency Virus type 1-infected individuals

Several reports have documented a better prognosis for HIV‐1‐infected patients co‐infected with GBV‐C, while other reports have contradicted such findings with the result that this issue remains controversial. We attempted to clarify the complicated status of the effect of GBV‐C co‐infection on HIV‐1‐infected patients. GBV‐C RNA was detected in 37 samples in 182 HIV‐1‐infected patients (20.3%) using RT/nested PCR. Of these, 3 were determined to be GBV‐C genotype 1, 12 were genotype 2, and the remaining 22 were genotype 3. The GBV‐C viral load quantified by real‐time PCR ranged from 7.8 × 10³ to 3.3 × 10⁶ copies/ml. Weakly negative correlation was observed between GBV‐C viral load and HIV‐1 viral load in 19 HAART‐naïve patients, indicating that a higher GBV‐C viral load is associated with a greater suppression of HIV‐1 replication. A previously published in vitro study suggested that GBV‐C infection would induce up‐regulation of RANTES, leading to suppression of HIV‐1 replication. However, in our present study, the blood RANTES level was significantly lower in the GBV‐C co‐infected group than in the uninfected group (190–9,959 vs. 264–31,038 pg/ml, P=0.004). Our results suggested that a suppression of HIV‐1 replication by GBV‐C co‐infection is not mediated by up‐regulated RANTES, and thus call for another as yet unknown factor.     

Discrimination of gamma-irradiated and nonirradiated Vibrio vulnificus by using real-time polymerase chain reaction

Aims: To develop a PCR strategy for Vibrio vulnificus in irradiated foods. Methods and Results: Real‐time PCR was used to discriminate between DNA from γ‐irradiated and nonirradiated cells. γ‐Irradiation at 1·08 KGy and above of cell suspensions containing 1 × 10⁶ CFU ml^?1 resulted in 0 CFU ml^?1. Real‐time PCR was able to detect 86·6% destruction by 1·08 KGy, while ethidium bromide monoazide (EMA) real‐time PCR was able to detect 93·2% destruction at this dose. With 3·0 and 5·0 KGy, EMA real‐time PCR was able to detect 99·3% and 100% destruction, respectively. Conclusions: The inability to detect via PCR extensively degraded DNA resulting from γ‐irradiation can be taken as evidence of cell death. The increased ability of EMA to further reduce the detectable number of target sequences via PCR, with DNA from cells exposed to increased doses of γ‐irradiation, can be considered to reflect the accompanying increase in membrane damage which allows EMA to penetrate the cells. Significance and impact of the study: This is the first study that has made use of the PCR to discriminate between γ‐irradiated and nonirradiated bacterial cells and has led to insights regarding the ability of the PCR to discriminate between nonirradiated and γ‐irradiation destroyed cells.     

Rapid Detection of Colletotrichum lagenarium,Causal Agent of Anthracnose of Cucurbitaceous Crops,by PCR and Real‐time PCR

Rapid and accurate polymerase chain reaction (PCR) and real‐time PCR methods were developed for the detection of Colletotrichum lagenarium, the causal agent of anthracnose, in tissues of squash (Cucurbita moschata), watermelon (Citrullus lanatus), cucumber (Cucumis sativus) and muskmelon (Cucumis melo). PCR assays amplified different internal transcribed spacer sequences from C. lagenarium, so effectively detected this pathogen in infected tissues. PCR analysis with the primer co‐m‐337F1/R1 was able to differentiate C. lagenarium from other fungal pathogens, including Colletotrichum spp., Fusarium spp., Alternaria spp. and Didymella spp. An optimized real‐time PCR assay was developed to detect and monitor C. lagenarium in both infected plant tissues and soil samples. The sensitivity of real‐time PCR can detect down to 1 pg of DNA. Thus, PCR‐based analysis is a useful technique for rapid detection and diagnosis of C. lagenarium in infected plants or infested soils.