Development of a TaqMan Real‐Time Polymerase Chain Reaction Assay for Detection and Quantification of Fusarium oxysporum f. sp. lycopersici in Soil

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is an important disease of tomato. Pathogenicity and vegetative compatibility tests, although reliable, are laborious for the identification of FOL isolates and cannot efficiently quantify population densities of FOL in the soil. The objective of this study was to develop a rapid, sensitive and quantitative real‐time polymerase chain reaction (PCR) assay for detecting and quantifying FOL in soil. An inexpensive and relatively simple method for soil DNA extraction and purification was developed based on bead‐beating and a silica‐based DNA‐binding method. A TaqMan probe and PCR primers were designed using the DNA sequence of the species‐specific virulence gene SIX1, which is only present in isolates of FOL, not in isolates of other formae speciales or non‐pathogenic isolates of F. oxysporum. The real‐time PCR assay successfully amplified isolates of three races of FOL used in this study and quantified FOL DNA in soils, with a detection limit of 0.44 pg of genomic DNA of FOL in 20 μl of the real‐time PCR. A spiking test performed by adding different concentrations of conidia to soil showed a significant linear relationship between the amount of genomic DNA of FOL detected by the real‐time PCR assay and the concentration of conidia added. In addition, the real‐time PCR assay revealed a significant quadratic regression for a glasshouse experiment between disease severity and DNA concentration of FOL. The soil DNA extraction method and real‐time PCR assay developed in this study could be used to determine population densities of FOL in soil, develop threshold models to predict Fusarium wilt severity, identify high‐risk fields and measure the impact of cultural practices on FOL populations in soils.     

Differences in parasitism of Meloidogyne incognita and two genotypes of M. arenaria on Solanum torvum in Japan

Two genotypes of root‐knot nematode, Meloidogyne arenaria (A2‐O and A2‐J), are found in Japan. They were distinguished from each other based on mitochondrial DNA sequences. The primer set (C2F3/1108) amplified a 1.7‐kb fragment from A2‐J, whereas a 1.1‐kb fragment was amplified from A2‐O. M. arenaria (A2‐O) was detected in local regions of southern Japan, whereas M. arenaria (A2‐J) was widespread from the Kyushu region to the Tohoku region. The distribution of M. arenaria (A2‐J) overlaps with the cultivation area of eggplant. Solanum torvum is used worldwide as a rootstock for eggplant cultivation, and it is resistant to Meloidogyne spp. In particular, it is reported that S. torvum is resistant to M. arenaria outside Japan. In this study, we inoculated S. torvum rootstock cultivars with M. arenaria (A2‐J), M. arenaria (A2‐O) and Meloidogyne incognita populations. Although M. incognita and M. arenaria (A2‐O) produced only a few egg masses on S. torvum, thereby confirming its resistance, the four geographical populations of M. arenaria (A2‐J) produced large numbers of egg masses on S. torvum. This study confirmed that S. torvum is resistant to M. incognita and M. arenaria (A2‐O) populations, but susceptible to populations of M. arenaria (A2‐J) in the eggplant production area of Japan.     

Occurrence of Meloidogyne spp. in Cerrado Vegetations and Reaction of Native Plants to Meloidogyne javanica

The Cerrado biome represents a hotspot of biodiversity. Despite this, the nematofauna in this biome has not been well characterized, especially that related to root‐knot nematodes. This work aimed to identify Meloidogyne species present in different cerrado vegetations and to investigate potential hosts of Meloidogyne javanica in this biome. Soil samples (250) were collected in native areas of cerrado vegetation located at the National Park of Brasília (PNB) (125 samples) and Água Limpa Farm (FAL) (125 samples), and transferred to sterile pots. Single tomato plants cv. Santa Clara (susceptible) were transplanted into individual pots and maintained for 90 days under glasshouse. Females of Meloidogyne spp. were extracted from tomato roots and identified based upon esterase phenotypes and confirmed with PCR using specific sequence characterized amplified regions (SCAR) primers. Native plants were inoculated with 10 000 individuals (eggs + J2) of a pure culture of M. javanica and maintained under glasshouse for 6 months. From the 250 samples collected, 57 (22.8%) presented Meloidogyne spp. A total of 66 Meloidogyne populations were identified as follows: M. javanica (75.76%), M. incognita (10.60%), M. hapla (9.1%), M. morocciensis (3.03%) and M. arenaria (1.51%). The following esterase phenotypes were detected: M. javanica (J3 and J2), M. incognita (I1 and I2), M. hapla (H1), M. morocciensis (A3) and M. arenaria (A2). The SCAR primers incK14F/incK14R, Fjav/Rjav and Fh/Rh amplified specific fragments in M. incognita (399 bp), M. javanica (670 bp) and M. hapla (610 bp) and can be used for identification of indigenous Meloidogyne spp. from cerrado. The primer set Far/Rar is not specific for M. arenaria due to the amplification of DNA in M. morocciensis. Mimosa caesalpiniifolia was the only native plant in which M. javanica developed a high reproductive rate, and it is probably a host for this nematode in cerrado.     

Evaluation of High‐resolution Melting Curve Analysis as a New Tool for Root‐knot Nematode Diagnostics

In the detection of plant pests, speed and accuracy are vital. High‐resolution melting curve (HRMC) analysis was therefore evaluated as a new tool for the identification of root‐knot nematodes (Meloidogyne spp.). On the basis of the second intergenic spacer (IGS2) region of the ribosomal DNA cistron, Meloidogyne chitwoodi, M. fallax and M. hapla were successfully distinguished from each other and the group of the three tropical species, M. incognita, M. arenaria and M. javanica. Conversely, it was shown that the IGS2 region is not suitable for the tropical species M. enterolobii (senior synonym of M. mayaguensis) as the amplification of multiple fragments of different lengths prevented a reliable HRMC analysis. However, the obtained results provide a proof of principle that HRMC analysis can be a suitable single‐tube assay for fast and accurate root‐knot nematode identification.     

Rapid diagnosis of the invasive species,Frankliniella occidentalis (Pergande): a species‐specific COI marker

The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is an invasive species and currently occurs in only a few areas in China. An easy, accurate and developmental‐stage independent method to identify F. occidentalis would be a valuable tool to facilitate pest management decision making and, more importantly, to provide an early warning so actions can be taken to prevent its introduction into non‐infested areas. Morphological identification of thrips adults and, to a lesser extent, of second‐stage larvae is the main method currently available to identify F. occidentalis. Molecular identification, however, can be easily carried out by a non‐thrips‐specialist with a little training. In this study, DNA sequence data [within the mitochondrial cytochrome oxidase I gene (COI)] and polymerase chain reaction (PCR) were utilized to develop a molecular diagnostic marker for F. occidentalis. A primer set and PCR cycling parameters were designed for the amplification of a single marker fragment (340 bp) of F. occidentalis COI mtDNA. Specificity tests performed on 28 thrips species, efficacy tests performed on five immature developmental stages as well as on male and female adults and tests on primer sensitivity all demonstrated the diagnostic utility of this marker. Furthermore, the primer set was tested on seventeen F. occidentalis populations from different countries and invaded areas in China and proved to be applicable for all geographic populations. It was used successfully to clarify the distribution of F. occidentalis in the Beijing metro area. These results suggested that this diagnostic PCR assay provides a quick, simple and reliable molecular technique for the identification of F. occidentalis.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

Molecular Typing of Cyst‐Forming Nematodes Globodera pallida and G. rostochiensis,Using Real‐Time PCR and Evaluation of Five Methods for Template Preparation

Globodera pallida and G. rostochiensis are two cyst‐forming nematodes known to infest potato crops, causing severe economic losses worldwide. In this study, a real‐time TaqMan PCR assay was developed and optimized for the simultaneous detection of G. pallida and G. rostochiensis. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Four different DNA extraction methods and one rapid crude template‐preparation procedure were compared in terms of extraction purity, efficiency for PCR applications, utility and cost. Extraction methods A and B included two commercially available kits that utilize silica columns and magnetic beads, respectively. Method C was based on DNA isolation using Chelex resin, and method D was a standard chemistry in‐house protocol. Procedure E included the direct use of crude mixture composed of disrupted cysts in Tris–EDTA buffer. The multiplex TaqMan PCR assay successfully discriminated the two nematode species from all reference cyst samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional (Co) PCR tests, the overall Dsp and Dse were lower and estimated at 94 and 87% for G. pallida, and 97 and 88% for G. rostochiensis, respectively. Spectrophotometric results showed that DNA extraction methods A, B and C yielded the purest DNA and gave the lowest mean C_t values as well as the most consistent results in Co PCR. Alternative crude preparation method E resulted in statistically similar and C_t values consistent with those obtained with methods A to C when tested by TaqMan PCR. The developed assay, using crude template‐preparation E, allows the simple, accurate and cost‐effective testing of a large number of cyst samples and can be applied in surveys and certification schemes.     

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.     

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

Aim: The aim of this study was to develop a multiplex real‐time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens. Methods and Results: A primer set and three species‐specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia‐associated non‐Erysipelothrix spp. bacterial isolates. Cross‐reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection. Conclusion: The multiplex real‐time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive. Significance and Impact of the Study: The developed real‐time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.     

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.     

A TaqMan PCR Method for Routine Diagnosis of the Quarantine Fungus Guignardia citricarpa on Citrus Fruit

With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real‐time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real‐time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi‐copy rDNA gene. Co‐amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real‐time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real‐time PCR methods showed that the TaqMan method was more sensitive.     

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

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

Limited mitochondrial DNA variation within South Africa's black rhino (Diceros bicornis minor) population and implications for management

The taxonomy of African black rhinoceros (Diceros bicornis) remains unresolved. Maintaining levels of genetic diversity and species rescue by reintroduction and restocking requires its resolution. We compared the sequences of the mitochondrial DNA (mtDNA) control region for a total of 101 D. bicornis from three subspecies: D. b. minor, D. b. michaeli and D. b. bicornis. A single unique haplotype was found within the 65 D. b. minor samples from KwaZulu‐Natal (KZN) Province, South Africa, 55 of which came from Hluhluwe‐iMfolozi Game Park (HiP) and Mkuzi Game Reserve (MGR) source populations. However, six different haplotypes were represented in eleven D. b. minor samples from Zimbabwe. Similarly, published autosomal microsatellite data indicate low levels of diversity within the KZN D. b. minor populations. The low levels of mtDNA diversity within the KZN metapopulation point to the possible need for genetic supplementation. However, there is a need to determine whether the low levels of genetic variation within KZN D. b. minor are a result of the recent bottleneck or whether KZN historically always had low diversity.     

Real‐time PCR Detection of Sorghum Ergot Pathogens Claviceps africana,Claviceps sorghi and Claviceps sorghicola

Sorghum ergot is a serious disease that has caused major losses in sorghum growing regions worldwide. Claviceps africana, originally reported from Zimbabwe, is now the most widely distributed species causing ergot in many countries including the United States of America, whereas both C. africana and Claviceps sorghi exist in India. A third species (Claviceps sorghicola) has been described causing sorghum ergot in Japan. As the three species show morphological similarities, a DNA‐based assay is desirable for rapid identification in cases where ergot‐infected sorghum is found by regulatory authorities. We designed PCR primers and probes from the intron 3 region of the β‐tubulin gene (for C. africana and C. sorghi) and the intron 4 region of EF‐1α (for C. sorghicola) and tested them by real‐time PCR with purified DNA and ergot samples from the field and greenhouse. The primer and probe sets specifically amplified DNA from the respective species with a detection limit of c. 1 pg DNA. Genomic DNA from six other Claviceps species did not amplify in any of the three ergot species‐specific assays. The assays we describe will provide useful tools for detecting sorghum ergot pathogens in seed and grain shipments and for determining which species are present in the samples, thereby aiding in the regulatory decision‐making process.     

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.     

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.     

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 .     

Specific detection of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8 with TaqMan® PCR

Aims: To develop a strain‐specific TaqMan^® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain‐specific sequence selected using REP‐PCR (repetitive extragenic palindromic‐polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10‐fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan^® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan^® PCR method is a strain‐specific real‐time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.     

LNA probes in a real‐time TaqMan PCR assay for genotyping of Giardia duodenalis in wastewaters

Aims: This study describes an approach for genotyping Giardia cysts obtained from wastewater treatment plants (WTPs) in Spain using real‐time PCR (qPCR) in combination with immunomagnetic beads. Methods and Results: A 50‐cycle amplification of a 74‐bp fragment of the Giardia beta‐giardin gene was adopted from a previous qPCR method. Additionally, two locked nucleic acid (LNA) probes were designed (LNA P434 P1 for assemblage A and LNA P434 H3 for assemblage B). All 16 wastewater samples analysed were positive with the immunofluorescence assay (IFA). Assemblage A was detected in all WTP samples using primer–LNA probe P434 P1 set. Giardia duodenalis identification was confirmed by PCR–RFLP analysis and sequencing of the β‐giardin gene in the water samples found positive by IFA and qPCR. Among the 16 assemblage A isolates that were sequenced, two subtypes were identified; 11 corresponded to the A2 subgenotype, whereas three corresponded to the subgenotype A3. A mixture of subgenotypes was found in the remaining two isolates. Conclusions: The newly developed qPCR assays were able to discern G. duodenalis assemblages A and B in wastewater. Significance and Impact of the Study: The real‐time PCR assays provided a rapid method for detection and one‐step genotyping of G. duodenalis from wastewater samples, and its application would contribute to understanding the distribution and abundance of G. duodenalis assemblages A and B in wastewater.     

Investigating the ecology and evolution of cryptic marine nematode species through quantitative real-time PCR of the ribosomal ITS region

The presence of morphologically similar but genetically distinct species has impacted biogeographical and ecological paradigms. In marine sediments, free‐living nematodes form one of the most abundant and diverse faunal groups. Inferring the importance of nematode diversity for ecosystem functioning requires species‐level identification, which is hampered by the lack of easily observable diagnostic characters and the presence of cryptic species. New techniques are urgently needed to adequately study the ecology and evolution of cryptic species. The aim of the present study was to evaluate the potential of a quantitative real‐time PCR (qPCR) assay using the internal transcribed spacer (ITS) region of the ribosomal DNA to detect and quantify cryptic species of the R. (P.) marina complex. All primer pairs proved to be highly specific, and each primer pair was able to detect a single juvenile in a pool of 100 nematodes. C_t values were significantly different between developmental stages for all species except for PmIII. Despite differences between developmental stages, a strong correlation was observed between the amount of extracted DNA and the number of nematodes present. Relative and absolute quantification estimates were comparable and resulted in strong positive correlations between the qPCR estimate and the actual number of nematodes present in the samples. The qPCR assay developed here provides the ability to quickly identify and quantify cryptic nematode species and will facilitate their study in laboratory and field settings.