Real‐time PCR for identification of the soybean aphid,Aphis glycines Matsumura

The soybean aphid (Aphis glycines Matsumura) is an economically significant pest in North America, causing extensive damage to soybean crops through direct feeding damage and disease transmission. If unchecked, this pest could cause billions of dollars of damage to soybean crops. Identification of the soybean aphid can be difficult due to its small size, complex life cycle and morphological plasticity. Generally, an expert is required to identify a specimen. Additionally, identification of some life stages, such as eggs, is impossible. DNA barcoding has been successfully used to differentiate aphid species, including A. glycines, based on sequencing of a standardized gene region. Although this method represents an important step towards accurate identification, samples must still be sent to specialized facilities for analysis. Using existing DNA barcode sequences in the publically accessible Barcode of Life Data System (BOLD; www.boldsystems.org ), species‐specific differences were identified and used to develop a real‐time PCR assay to identify soybean aphids. This assay can be run on portable systems for rapid, accurate and simple identification in the field. The use of a non‐destructive DNA extraction protocol allows the original insect to be vouchered and therefore available for further study if necessary. This work represents an important step in soybean aphid management.     

A Real‐time PCR Assay to Identify Meloidogyne minor

Meloidogyne minor is a small root‐knot nematode that causes yellow patch disease in golf courses and severe quality damage in potatoes. It was described in 2004 and has been detected in The Netherlands, England, Wales, Northern Ireland, Ireland and Belgium. The nematode often appears together with M. naasi on grasses. It causes similar symptoms on potato tubers as M. chitwoodi and M. fallax, which are both quarantine organisms in Europe. An accurate identification method therefore is required. This study describes a real‐time PCR assay that enables the identification of M. minor after extraction of nematodes from soil or plant samples. Alignments of sequences of rDNA‐ITS fragments of M. minor and five other Meloidogyne species were used to design a forward primer Mminor_f299, a specific primer Mminor_r362 and the specific MGB TaqMan probe P_Mm_MGB321. PCR with this primers and probe results in an amplicon of 64 bp. The analytical specificity of the real‐time PCR assay was assessed by assaying it on six populations of M. minor and on 10 populations of six other Meloidogyne species. Only DNA from M. minor gave positive results in this assay. The assay was able to identify M. minor using DNA from a single juvenile independent from the DNA extraction method used.     

Development of novel real‐time TaqMan® PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real‐time polymerase chain reaction assays using fluorescently labelled TaqMan^® MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost‐saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.     

A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.     

Real‐time PCR identification of lake whitefish Coregonus clupeaformis in the Laurentian Great Lakes

The purpose of this study was to develop a real‐time PCR assay to specifically identify lake whitefish Coregonus clupeaformis in larval fish assemblages based on a 122 bp amplicon from the mitochondrial genome. The efficiency of the reaction, as calculated from the standard curve, was 90·77% with the standard curve having an r² value of 0·998. Specificity of the assay provided single melt peak in a melt‐curve analysis and amplification of only the target species. The assay successfully identified target DNA in as low as 0·1% proportion of a DNA mixture. This assay was designed on the portable Smart Cycler II platform and can be used in both field and laboratory settings to successfully identify C. clupeaformis.     

Development and evaluation of real‐time PCR assays for bloodmeal identification in Culicoides midges

Culicoides (Diptera: Ceratopogonidae) midges are the biological vectors of a number of arboviruses of veterinary importance. However, knowledge relating to the basic biology of some species, including their host‐feeding preferences, is limited. Identification of host‐feeding preferences in haematophagous insects can help to elucidate the transmission dynamics of the arboviruses they may transmit. In this study, a series of semi‐quantitative real‐time polymerase chain reaction (qPCR) assays to identify the vertebrate host sources of bloodmeals of Culicoides midges was developed. Two pan‐reactive species group and seven species‐specific qPCR assays were developed and evaluated. The assays are quick to perform and less expensive than nucleic acid sequencing of bloodmeals. Using these assays, it was possible to rapidly test nearly 700 blood‐fed midges of various species from several geographic locations in Australia.     

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.     

A real‐time PCR assay for the differentiation of Candida species

Aims: We established a real‐time PCR assay for the detection and strain identification of Candida species and demonstrated the ability to differentiate between Candida albicans the most common species, and also Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida dubliniensis by LightCycler PCR and melting curve analysis. Methods and Results: The DNA isolation from cultures and serum was established using the QIAmp Tissue Kit. The sensitivity of the assay was ≥ 2 genome equivalents/assay. It was possible to differentiate all investigated Candida species by melting curve analysis, and no cross‐reaction to human DNA or Aspergillus species could be observed. Conclusions: The established real‐time PCR assay is a useful tool for the rapid identification of Candida species and a base technology for more complex PCR assays. Significance and Impact of the Study: We carried out initial steps in validation of a PCR assay for the detection and differentiation of medically relevant Candida species. The PCR was improved by generating PCR standards, additional generation of melting curves for species identification and the possibility to investigate different specimens simultaneously.     

Real‐time PCR for Helicobacter pylori diagnosis. The best tools available

Background

Knowledge of antimicrobial susceptibility, especially to macrolides, has become crucial for the management of Helicobacter pylori infection. Our aim was to evaluate two new PCR kits able to detect H. pylori in gastric biopsies as well as the mutations associated with macrolide resistance.

Materials and Methods

Two hundred successive biopsies (received from gastroenterologists all over France) were used. The two new kits tested were Amplidiag H. pylori+Clari^R from Mobidiag Espoo, Finland, and RIDA^®GENE H. pylori from R‐Biopharm, Darmstadt, Germany. Culture and a validated in‐house real‐time PCR were also performed, and in the case of a positive culture, Etest for clarithromycin was carried out. Discrepancies were solved by looking at the pathologic data.

Results

Culture was positive in 68 cases (34%), and with our in‐house real‐time PCR in these 68 cases plus 5 others (N = 73, 36%). All were also detected by the two new kits. In addition, RIDA^®GENE H. pylori detected one more positive also detected by Amplidiag H. pylori+Clari^R, and Amplidiag detected two other positives. Of these three additional cases, pathology confirmed the positivity for two. Only one case diagnosed by Amplidiag could be considered as a false positive. With regard to clarithromycin resistance, 22 cases were detected. The corresponding mutations (A2142/43G) were all identified with the three PCRs.

Conclusions

These two new kits which have an excellent sensitivity and specificity are convenient to use, adaptable to different thermocyclers, provide quick results, and deserve to be used in H. pylori diagnosis for a better choice of treatment regimen.     

Real‐time PCR detection of Enterococcus faecalis associated with amyloid arthropathy

Aims: To develop a RT‐PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and Results: Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT‐PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single‐nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used. Conclusions: The real‐time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis. Significance and Impact of the Study: The new method allows a rapid screening of isolates with results available after only few hours. This RT‐PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.     

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.     

A new set of PCR assays for the identification of multiple human adenovirus species in environmental samples

Aims:  To assess human adenovirus (HAdV) diversity in environmental samples based on sequence comparisons of hexon gene fragments amplified using newly designed HAdV-specific polymerase chain reaction (PCR) assays.
Methods and Results:  Six PCR primer sets were designed based on 56 aligned hexon sequences from NCBI GenBank to amplify different hexon gene sections (241–349 bp) of the six HAdV species. The amplified hexon genes from wastewater samples were cloned, sequenced, and compared with those in publicly accessible databases (i.e. NCBI GenBank) by using the B last program. A total of 46 analysed positive clones were affiliated to five HAdV serotypes, i.e. 1, 2, 12, 31 and 41. Similarities between the cloned and database hexon sequences ranged from 95·9 to 100% (with an average of 98·1 ± 1·0%).
Conclusion:  The designed primers showed higher amplification efficiencies when compared with the existing assays. Using the new assays, HAdV species A, C, and F (serotypes 1, 2, 12, 31 and 41 in particular) were identified in the studied municipal wastewater.
Significance and Impact of the Study:  The six PCR primer sets developed in this study can be used to efficiently amplify hexon gene fragments in HAdV. Multiple HAdV serotypes identified in the municipal wastewater provide new information about HAdV diversity in environmental samples.     

Real‐time PCR identification of the ambrosia beetles,Trypodendron domesticum (L.) and Trypodendron lineatum (Olivier) (Coleoptera: Scolytidae)

The European hardwood ambrosia beetle (Trypodendron domesticum) and the striped ambrosia beetle (Trypodendron lineatum) are wood‐boring pests that can cause serious damage to lumber, resulting in a need for management of these pests in logging and lumber industries. Natural populations of ambrosia beetles exist throughout the world, but movement of ambrosia beetles into new habitats, particularly via international trade, can result in the establishment of invasive species that have the potential to spread into new territory. Efforts to monitor ambrosia beetle populations are time‐consuming and could be greatly enhanced by the use of molecular methods, which would provide accurate and rapid identification of potentially problematic species. Here, we present new real‐time PCR assays for the detection and identification of T. domesticum and T. lineatum. The methods described herein can be used with a variety of sampling strategies to enable timely and well‐informed decision‐making in efforts to control these ambrosia beetles.     

Real‐time PCR quantification of Bemisia tabaci (Homoptera: Aleyrodidae) B‐biotype remains in predator guts

The cotton whitefly, Bemisia tabaci (Gennadius) B‐biotype, is fed on by a wide variety of generalist predators, but there is little information on these predator–prey interactions, especially under field conditions. In this study, a real‐time polymerase chain reaction (PCR) assay was developed to quantify B. tabaci B‐biotype remains in predator gut. The B. tabaci B‐biotype genomic DNA copy number was referred to the actual amount of BT1 isolate, the B. tabaci B‐biotype specific DNA fragment. The numbers of BT1 isolate in one B. tabaci B‐biotype egg, individual adult and a single red‐eyed nymph were 2.56 × 10³, 2.56 × 10⁴, and 1.29 × 10⁴ copies, respectively. When Propylaea japonica adults fed on one, two, four, eight or 16 red‐eyed nymphs, the detected numbers of BT1 isolate ranged from 2.77 × 10⁴ to 4.05 × 10⁵ copies, forming a strong linear relationship (R² = 0.9899). Following the consumption of two red‐eyed nymphs, prey DNA was detectable in 100% of P. japonica at t = 0, decreasing to 80.0% and 60.0% after 1–4 h and 8 h of digestion, respectively, with 3.36 × 10⁴–1.25 × 10³ BT1 isolate copies. The predation by field‐collected predators, 26 larvae of P. japonica, and of Harmonia axyridis each, Chrysopa spp. larvae (Chrysopa pallens and C. formosa, 18 individuals in total), and a single adult of Scymnus hoffmanni, 19 adults of Orius sauteri and nine adult spiders (Erigonnidium graminicolum and Neoscona doenitzi), on B. tabaci B‐biotype were quantified. Of the 99 analysed predator individuals, 3.65 × 10²–4.60 × 10⁵ copies of BT1 isolate, equivalent to 0.8–18.8 red‐eyed nymphs were detected. These results suggest that TaqMan real‐time PCR technology may provide a rapid and sensitive method for quantifying B. tabaci B‐biotype remains in predator guts and will be invaluable in assessing the food web relationship between prey and arthropod predators.     

Real‐time PCR validation of visually identified snapper Chrysophrys auratus (Sparidae) eggs

In this study, a total of 212 eggs were visually identified as snapper Chrysophrys auratus. Real‐time PCR confirmed visual identification in 69% of cases but corroboration varied widely among plankton samples. The use of molecular tools to support visual identification prior to adopting daily egg production stock assessment methods should be considered.