Species-specific Primer Sets Research Articles

Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis

Powdery mildew and downy mildew are two devastating diseases on cucumber and other cucurbit crops caused by Podosphaera xanthii and Pseudoperonospora cubensis, respectively. Identification and detection of these pathogens from field and plant material could be significant for the selection of resistant varieties and formulation of disease management strategies. In the present study, a duplex qPCR assay developed for simultaneous detection and quantification of both pathogens from different samples. Two sets of species-specific primers developed for the detection of P. xanthii and P. cubensis pathogens by targeting the internal transcribed spacer (ITS) region of the rDNA gene cluster. The specificity of designed primers was also evaluated against the different microbial, plant, soil, and environmental samples. Initially, the individual assays for P. cubensis and P. xanthii were validated using their corresponding species-specific primers, which amplified the prominent and distinctive products of ~ 705 bp and ~ 290 bp size, respectively. SYBR green-based duplex real-time PCR assay was developed to detect and quantify both mildew pathogens from different field samples. The species-specific oligonucleotide primer sets showed high specificity with melt curve peaks at 85.83 °C and 88.05 °C, for P. xanthii and P. cubensis, respectively. The relative quantification and lowest detection limit of qPCR assays using tenfold diluted plasmid (Csp1 and Csd1) DNA were estimated (0.1 pg/µl) through a standard curve. In this study, the species-specific PCR and qPCR assays in both simplex and duplex formats have been validated successfully. These assays could be useful for efficient detection and quantification of mildew pathogens from the cucumber and other cucurbit crops.

Characterizing the spatial and temporal occurrence patterns of the endangered botiid loach Parabotia curtus by environmental DNA analysis using a newly developed species-specific primer set

We report a novel species-specific primer set for detection of the endangered botiid loach Parabotia curtus in environmental waters. A primer test using DNA extracted from tissues of P. curtus and four other loach species indicated its high specificity to P. curtus. Application of the primer set to field samples collected throughout the year in a natural habitat confirmed its ability to characterize spatial and temporal occurrence patterns of this rare, small fish that often inhabits turbid waters.

A novel environmental DNA detection approach for the wading birds Platalea leucorodia, Recurvirostra avosetta and Tringa totanus

Wading birds play an important role in coastal and wetland ecosystems. As a result of anthropogenic disturbances, numbers of wading birds have declined over the past years, and therefore, the monitoring of bird species is crucial to preserve their habitats and counteract this. Due to known limitations of conventional monitoring, molecular approaches are increasingly becoming complementary methods. Thus, we evaluated the potential of an environmental DNA (eDNA) monitoring approach for wading birds. We developed three species-specific primer sets for wading birds targeting the COI region. After examining the primers in silico and on extracted bird DNA, the primers were used to successfully amplify eDNA from water samples.

The effect of temperature on environmental DNA degradation of Japanese eel

AbstractThe environmental DNA (eDNA) technique is a convenient and powerful tool to detect rare species. Knowledge of the degradation rate of eDNA in water is important for understanding how degradation influences the presence and/or estimate biomass of aquatic animals. We developed a new set of species-specific primers and probe to detect eDNA of Japanese eel Anguilla japonica, which is a commercially important and endangered species, and then conducted a laboratory experiment to quantify the temperature-dependent degradation of emitted eDNA. Eels were held in tanks at five different temperature levels from 10 to 30 °C and water from each tank was sampled and kept in bottles at each temperature over 6 days. The concentration of eDNA was measured every day and the results showed that temperature (T) had a significant and positive effect on the degradation rate (k) as k = 0.02T + 0.18. Improved understanding of the effect of temperature on degradation rates would help data interpretations and adjustments would increase the reliability of eDNA analysis in future studies.

Genome-based species-specific primers for rapid identification of six species of Lactobacillus acidophilus group using multiplex PCR.

Many Lactobacillus species are frequently isolated from dairy products, animal guts, and the vaginas of healthy women. However, sequencing-based identification of isolated Lactobacillus strain is time/cost-consuming and lobor-intensive. In this study, we developed a multiplex PCR method to distinguish six closely related species in the Lactobacillus acidophilus group (L. gasseri, L. acidophilus, L. helveticus, L. jensenii, L. crispatus, and L. gallinarum), which is based on species-specific primer sets. Altogether, 86 genomes of 9 Lactobacillus species from the National Center of Biotechnology Information (NCBI) database were compared to detect species-specific genes and design six species-specific primer sets. The PCR conditions of the individual primer sets were optimized via gradient PCR methods. A final multiplex PCR condition was also optimized for a mixture of all six primer sets mixed. When identifying a single strain, the optimized multiplex PCR method can specifically detect one of the six species, but no band was amplified at least from the other Lactobacillus and Enterococcus species. These results indicated that species-specific primer sets designed from the genome comparison could identify one strain within the six Lactobacillus species by a single PCR reaction. Using the method described here, we will be able to save time, cost, and labor during species identification and screening of commercially important probiotic lactobacilli.

Environmental DNA and Specific Primers for Detecting the Invasive Species Ectopleura crocea (Hydrozoa: Anthoathecata) in Seawater Samples

In marine environments, environmental DNA (eDNA) can be effectively detected and possibly quantified when combined with molecular techniques, as demonstrated by several recent studies. In this study, we developed a species-specific primer set and a probe to detect the distribution and biomass of an invasive hydrozoan in South Korea, Ectopleura crocea. These molecular markers were designed to amplify a 187 bp region based on mitochondrial cytochrome c oxidase subunit I (COI) of E. crocea and were tested on seawater samples from 35 Korean harbors in 2017. Of the 35 sites we investigated, only nine harbors returned positive detections when using traditional survey methods, while surveys based on the use of eDNA techniques detected E. crocea DNA in all seawater samples. These results suggest that eDNA surveys based on molecular techniques are more effective at identifying species distribution and estimating biomass than traditional surveys based on visual assessment of morphology.

Evaluation of host-based molecular markers for the early detection of human sepsis

We have identified 24 molecular markers, based on circulating nucleic acids (CNA) originating from the human genome, which in combination can be used in a quantitative real-time PCR (qPCR) assay to identify the presence of human sepsis, starting two to three days before the first clinical signs develop and including patients who meet the SEPSIS-3 criteria. The accuracy was more than 87 % inside of the same patient cohort for which the markers were developed and up to 81 % in blind studies of patient cohorts which were not included in the marker development. As our markers are host-based, they can be used to capture bacterial as well as fungal sepsis, unlike the current PCR-based tests, which require species-specific primer sets for each organism causing human sepsis. Our assay directly uses an aliquot of cell-free blood as the substrate for the PCR reaction, thus allowing to obtain the diagnostic results in three to four hours after the collection of the blood samples.

Identification of Heterodera glycines (Tylenchida; Heteroderidae) Using qPCR.

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea’s soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R2 = 0.996, slope = −3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

Multiplex PCR assay to identify Pseudomonas grimontii and P. marginalis simultaneously

A multiplex colony-direct PCR method was developed to identify Pseudomonas grimontii and P. marginalis simultaneously. Species-specific primer sets were designed based on the sequences of the rpoD or rpoB genes. The universal primer set for the 16S rRNA gene, chosen as an internal control, was also added to the PCR mixture to avoid incorrect judgement due to false negatives. By this method, two DNA fragments, one from the species-specific amplifications and the other from the 16S rRNA gene, were stably obtained from the strains belonging to the targeted species, showing that this method is useful for their identification.

Evaluation of white Kwao Krua (Pueraria candollei Grah. ex Benth.) products sold in Thailand by molecular, chemical, and microscopic analyses.

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.

Biomass-dependent emission of environmental DNA in jack mackerel Trachurus japonicus juveniles.

Environmental DNA (eDNA) from juvenile jack mackerel Trachurus japonicus was detected in tanks with 1, 3, 10, or 30 individuals per tank. Quantitative PCR using a set of species-specific primers and a probe revealed that the concentration of eDNA increased almost linearly with the density of fish. The coefficient of determination (R2 ) in the linear regression was lower than values previously reported for freshwater fishes in similar settings.

Development and validation of ultrafast PCR assays to detect six species of edible insects

With the increasing use of insects as a food source, specific and sensitive detection methods for edible insects are required. To detect six insects (silkworm, mealworm, two-spotted cricket, rice grasshopper, white-spotted flower chafer beetle larvae, and rhinoceros beetle larvae) that have been approved for food consumption in Korea, an ultrafast PCR system was developed based on a microfluidic chip. Insect species-specific primer sets for the identification of the six edible insects were selected targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene, and estimated for their specificity against 10 species of insects, which amplified only target insect species. The sensitivity of each primer set was independently validated in triplicate and was as little as 1 pg of DNA extracted from all insects. A total of 16 commercially processed products were used to verify the availability of these assays and to monitor the presence of labeled insect species. The ultrafast PCR assays developed in this study take approximately 20 min, and additionally confirm target species through post-PCR melt curve analysis. Thus, six species of edible insects could be rapidly and accurately identified in commercial food products using specific and sensitive ultrafast PCR methods.

Development of DNA-based species-specific real-time PCR markers for four berry fruits and their application in commercial berry fruit foods

Berry fruits have attracted attention because of their purported benefits for aging, cardiovascular disease, and cancer. Therefore, highly priced berry fruits might be targets for food adulteration and fraud. In this study, eight species-specific primer sets based on the single nucleotide polymorphism of the chloroplast genomes of four berry fruits (aronia, blackberry, cranberry, and strawberry) were developed for quantitative real-time PCR (qPCR) analysis by SYBR Green staining with the aim of preventing berry fruit food fraud. The developed primer pairs exhibited high efficiencies ranging from 88 to 110% with strong correlation coefficients (R2 > 0.99) for the amplification of each target species. However, no clear correlation coefficients were evident for non-target species. To evaluate the practicality of the developed primers, 18 commercial berry fruit products were verified by qPCR analysis. The developed primer pairs were amplified to a low Ct value (range 16.1–23.3) for the target species and proved capable of detecting target species in berry fruit commercial foods. Therefore, the developed qPCR-based species-specific markers could be suitable for the prevention of berry fruit food fraud and to verify marker reliability.

Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene.

Talaromyces species are typical fungi capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Hydrophobins, which are unique to fungi, are small secreted proteins that form amphipathic layers on the outer surface of fungal cell walls. In this study, species-specific primer sets for detecting and identifying Talaromyces macrosporus and Talaromyces trachyspermus were designed based on hydrophobin gene sequences. A conventional polymerase chain reaction (PCR) assay using these primer sets produced species-specific amplicons for T. macrosporus and T. trachyspermus. The detection limit for each primer set was 100pg template DNA. This assay also detected fungal DNA extracted from blueberries inoculated with T. macrosporus. Other heat-resistant fungi, including Byssochlamys, Neosartorya and Talaromyces species, which cause food spoilage, were not detected in PCR amplifications with these primer sets. Furthermore, a conventional PCR assay using a crude DNA extract as the template also yielded amplicons specific to T. macrosporus and T. trachyspermus. The simple and rapid PCR assay described herein is highly species-specific and can reliably detect T. macrosporus and T. trachyspermus, suggesting it may be relevant for the food and beverage industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The heat-resistant ascospores of Talaromyces macrosporus and Talaromyces trachyspermus are responsible for food spoilage after pasteurization. Traditional methods for detecting fungal contamination based on morphological characteristics are time-consuming and exhibit low sensitivity and specificity. In this study, a conventional polymerase chain reaction (PCR) assay based on hydrophobin gene sequences was developed for the specific detection of T. macrosporus and T. trachyspermus. This detection method was simple, rapid and highly specific. These results suggest that the conventional PCR assay developed in this study may be useful for detecting T. macrosporus and T. trachyspermus in raw materials and processed food products.

Polymerase chain reaction assay targeting nox gene for rapid identification of Brachyspira canis in dogs.

Genus Brachyspira, as Gram negative anaerobic bacteria, colonize in dogs intestine. The aim of the current study was to determine the prevalence of Brachyspira spp. for the first time in Iran and rapid identification of Brachyspira spp. in dogs by a new designment of a species-specific primer set for B. canis. One hundred fifty-one fecal samples were obtained from dogs by rectal swab. Twenty dogs suffered from diarrhea and 131 of them were healthy. In 9.27% (14/151) of samples, spirochaetes were detected on primary cultures by weak hemolysis and positive Gram staining and then Brachyspira genus was confirmed by NADH oxidase (nox) gene via polymerase chain reaction. Among 14 isolates, twelve isolates were B. canis, one isolate was B. intermedia and another one was non-typeable. From 12 B. canis, only eight isolates were detected by designed specific primers. Ten Brachyspira spp. were isolated from dogs ≤ 1 year old (10/67, 14.92%) and 4 isolates were from > 1 year old dogs (4/84, 4.76%). The isolation rates from healthy and diarrheic dogs were (12/131, 9.16%) and (2/20, 10.00%), respectively. A statistically significant association was observed between the presence of Brachyspira spp. and the age under one year. Based on our findings, the nox gene in B. canis might have more sequence variability compared to other Brachyspira spp.

PCR-based species identification applied in Japanese pear orchards to survey seasonal proportion changes of phytoseiid mite species

For this study, we developed a PCR-based method to identify the ribosomal internal transcribed spacer (ITS) sequences of six phytoseiid mite species found in Japanese pear orchards: Neoseiulus californicus (McGregor), Neoseiulus womersleyi (Schicha), Neoseiulus makuwa (Ehara), Amblyseius eharai Amitai and Swirski, Gynaeseius liturivorus (Ehara), and Amblyseius swirskii Athias-Henriot. Using the method, we can show seasonal proportion changes of phytoseiid mite species composition in Japanese pear orchards. Genomic DNA extracted individually from phytoseiid mites collected in the orchards are subjected to PCR of the ITS sequences using a universal primer set of which nucleotide sequences are conserved among the six phytoseiid mite species. Then, DNA samples showing PCR products with the universal primer set are used for another PCR of the ITS sequences using species-specific primer sets designed for respective phytoseiid mite species. Species-specific PCR is conducted in order of decreasing dominance of phytoseiid mite species. The method, which specifically identifies the six phytoseiid mite species irrespective of their sexes and developmental stages, might be useful for researchers who are lacking adequate morphological identification skills and nucleotide sequencing systems at their institutions.

Trichothecene Genotypes of Fusarium graminearum Populations Isolated from Winter Wheat Crops in Serbia.

Fusarium graminearum as the main causal agent of Fusarium head blight (FHB) and its ability to produce trichothecenes was investigated by molecular techniques. A total of 37 strains isolated from the wheat, harvested in Serbia in 2005, 2008 and 2015, and previously designated by morphological observation as F. graminearum, were used for trichothecene genotypes characterization. The strains were identified using the species-specific primer set FG16R/FG16F while genotypic characterization was done using specific TRI13 and TRI3 sequences of the trichothecene gene clusters. The PCR assays identified all strains as species of F. graminearum sensu stricto with the DON/15-ADON genotype. The quantification of the mycotoxin (DON) was performed using the biochemical assay. The high levels of DON (>20,000 µg kg−1) were recorded in all of the strains from 2005, four strains from 2008 and two strains from 2015. Weather data of the investigated seasons, showed that the optimal temperature, frequent rains and high relative humidity (RH) was very favourable for the development of F. graminearum, affecting the DON biosynthesis.

Comparison of oral flora before and after triple therapy for Helicobacter pylori eradication in patient with gastric disease.

The oral cavity is recognized as a major route for infection by Helicobacter pylori, which colonizes the gastric mucosa. Therapeutic options for elimination in patients with digestive disease have been established, though whether antibiotics are effective for H. pylori harbored in the oral cavity as well as oral commensal bacteria remain unknown. A 29-year-old woman visited a gastrointestinal clinic with a chief complaint of stomach ache. Gastroscopy and urine test findings for H. pylori IgG led to a definitive diagnosis of goose bumps gastritis induced by bacteria in the stomach, and triple therapy for H. pylori eradication was administered. Oral samples were obtained 10 times at a dental hospital clinic from 1 week before until 6months after starting triple therapy. Nested PCR amplifying the ureA gene and PCR assays using species-specific primer sets were performed to detect H. pylori and major oral commensal bacterial species, respectively, in those samples. Bacterial DNA encoding the ureA gene of H. pylori in oral specimens was detected prior to starting therapy, which was then reduced during and not detected after finishing treatment. Although the populations of major oral pathogenic bacteria, such as periodontopathic and oral streptococcal species, were drastically reduced during triple therapy, most had recovered within approximately 1 week after ending treatment. These results suggest that a conventional triple therapy approach for eradication of H. pylori in cases of digestive disease is effective for elimination of the bacterium from the oral cavity. On the other hand, after finishing such therapy, the oral flora composition is not drastically changed.

Detection of two Microsporidia pathogens of the European honey bee Apis Mellifera (Insecta: Apidae) in Western Siberia.

Two species of microsporidia, Nosema apis and Nosema ceranae, occur regularly and cause significant losses in apiculture throughout the world. N. ceranae is thought to be an emerging pathogen of the European honey bee which is replacing N. apis. Microscopic analysis of honey bees collected in Tyumen region, South-Western Siberia, suggested presence of two microsporidial pathogens slightly differing in spore size and shape. PCR detection using species-specific primer sets 312APIS and 218MITOC followed by PCR product sequencing confirmed the diagnosis of N. apis and N. ceranae, respectively. Microsporidia were present in private apiaries through 2008-2010, and among 21 colonies from 7 localities, two colonies were infected with both pathogens, while infections with N. apis only were detected in 8, and with N. ceranae only in 13 colonies. These data suggest that N. ceranae is widely spread in South-Western Siberia alongside with N. apis and is able to persist in the regions with average January temperatures below -18°C.

Markers for distinguishing Orostachys species by SYBR Green-based real-time PCR and verification of their application in commercial O. japonica food products

Human consumption of plant functional foods has been rapidly increasing owing to the health benefits they provide. In particular, in Korea, the plant Orostachys japonica has attracted attention for its anticancer and other effects. Of the 12 established Orostachys species, only three (viz., O. iwarenge, O. malacophyllus, and O. japonica) have been allowed for use as foods in Korea. In this study, 12 species-specific primer sets based on single nucleotide polymorphisms of five chloroplast genes and one nuclear gene were developed to discriminate Orostachys species through quantitative real-time PCR (qPCR) analysis with SYBR Green staining. The efficiencies of the designed primer pairs in amplifying the target species ranged from 80 to 110%, with strong correlation coefficients (R2 > 0.99), whereas no clear correlation coefficient was evident for the non-target species. In order to verify the specificity of the 12 developed Orostachys-specific primers, binary mixtures of the DNAs (tenfold serially diluted samples) from the target species and each of the other non-target species were generated for qPCR analysis, with results suggesting that the primers could clearly discriminate at least 0.1% of O. japonica DNA (10 pg) in the mixtures. With regard to the feasibility of the developed qPCR system for detecting Orostachys species in O. japonica food products, O. japonica DNA was detected in all eight commercial products tested, with low Ct values (< 20), whereas none of the other Orostachys species DNAs were detected, confirming that the tested foods contained only O. japonica. Therefore, developed primers and qPCR conditions would be useful for verifying the authenticity of commercial O. japonica food products.