Species-specific Probes Research Articles

Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species.

With the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato-Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.

Genetic diversity and prevalence of piroplasm species in equids from Turkey.

Equine piroplasmosis (EP) is a protozoon disease caused by Babesia caballi and Theileria equi transmitted by ticks from the Ixodidae family. This study investigated the genetic heterogeneity and diversity of piroplasm genotypes using the Reverse Line Blotting (RLB) technique for piroplasm species in equids in Turkey. A total of 233 blood samples from 142 horses and 91 donkeys were collected in Şanlıurfa, Tunceli, and Iğdır. The RLB assay was performed for simultaneous detection of piroplasm species. The prevalence of piroplasm infection was 33.5% (95% CI; 27.4-39.9). T. equi was the most common species (%20.2; 95% CI 15.2-25.9), whereas B. caballi infection was not detected. Nine samples hybridized to the Theileria/Babesia and Theileria catch-all probes but did not hybridize to any species-specific probe tested, suggesting the presence of unrecognized Theileria variants or genotypes. Nine samples that gave Theileria spp. signal and two samples that gave T. equi specific signal were sent for sequence analysis and the A and D genotypes of T. equi were found. In addition, T. annulata, B. ovis, and B. canis species were detected in the samples and verified by sequence analysis in Turkey. More research is needed to understand the clinical impact of atypical piroplasm infections in equids.

Oral microbiome development during childhood: an ecological succession influenced by postnatal factors and associated with tooth decay.

Information on how the oral microbiome develops during early childhood and how external factors influence this ecological process is scarce. We used high-throughput sequencing to characterize bacterial composition in saliva samples collected at 3, 6, 12, 24 months and 7 years of age in 90 longitudinally followed children, for whom clinical, dietary and health data were collected. Bacterial composition patterns changed through time, starting with "early colonizers", including Streptococcus and Veillonella; other bacterial genera such as Neisseria settled after 1 or 2 years of age. Dental caries development was associated with diverging microbial composition through time. Streptococcus cristatus appeared to be associated with increased risk of developing tooth decay and its role as potential biomarker of the disease should be studied with species-specific probes. Infants born by C-section had initially skewed bacterial content compared with vaginally delivered infants, but this was recovered with age. Shorter breastfeeding habits and antibiotic treatment during the first 2 years of age were associated with a distinct bacterial composition at later age. The findings presented describe oral microbiota development as an ecological succession where altered colonization pattern during the first year of life may have long-term consequences for child´s oral and systemic health.

Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique

Koumiss is a popular dairy product in many lands, traditionally prepared from mare milk with spontaneous fermentation. Mare milk and its fermented derivates are more expensive than cow milk and its fermented derivates, and the possibility exists for producers and dealers to adulterate equine products with bovine items. In this work, we described the development of a triplex real-time PCR based on species-specific TaqMan probes for identification of bovine and equine DNA in milks and dairy products. In addition, a novel designed endogenous control was simultaneously amplified to eliminate possible false negatives. With this methodology, bovine and equine DNA were specifically identified by employing developed primers and probes. The limits of detection of this method were 0.001 ng for cow milk, yogurt, and mare milk, and 0.005 ng for sour soup and koumiss, respectively. In addition, the triplex real-time PCR assay for authentication of animal-derived products was effectively validated using binary DNA and milk mixtures, exhibiting well in terms of specificity, sensitivity, and reproducibility. In short, the triplex PCR assay was verified to be a time-saving and money-saving technique for the identification of bovine and equine DNA in milks and dairy products.

Inferring the molecular affinity of Indian pangolin with extant Manidae species based on mitochondrial genes: a wildlife forensic perspective

Pangolins are the world`s most trafficked mammalian species classified under family Manidae and face severe threat of extinction, largely due to the illicit trade of its parts and products, especially scales, in international markets. Pangolin scales are believed to be used in Traditional Chinese Medicines (TCM) and meat is used as delicacies in restaurants. Of the eight extant species of pangolin, morphological discrimination is easy but the situation becomes precarious once the scales and meat samples are seized and it is difficult to identify species based on morphology in such cases. However, wildlife DNA forensics has played an instrumental role in the identification of species from such type of materials.The present study investigated that three mitochondrial genes (Cyt b, 16S rRNA, and 12S rRNA) clearly showed the variation among seven extant pangolin species (Manis culionensis; possibly extinct), whereas, maximum variation was obtained in cytochrome b when compared to another two mitochondrial genes.The present study revealed that obtained SNPs based on short sequence length (Intervals) within the three mitochondrial genes will be helpful to design the short molecular marker and species-specific probe that is used in wildlife forensic for identifying pangolin species from the degraded sample. We also advocate using more than one molecular marker for species discrimination so as to minimize any false identification of the mammal's species reported in the trade. Furthermore, data generated from the study would help in strengthening the DNA database of Indian pangolin species.

Molecular Evidence for a Novel Species of Babesia in Unfed Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

This study reports a novel species of Babesia in unfed Rhipicephalus sanguineus sensu lato and its phylogenetic relationship to other species. One-hundred and forty DNA samples extracted from tick pools comprising 5,403 unfed R. sanguineus s.l. ticks were screened for Babesia. Overall Babesia infection was detected in female, male, and nymph pools with an infection rate maximum likelihood estimate (MLE) of 1.98 (CI 0.65-4.74), 0.50 (CI 0.03-2.40), and 0.07 (CI 0.01-0.21), respectively. Three adult female tick pools (MLE 1.44, CI 0.38-3.88) and one adult male pool (MLE 0.50, CI 0.03-2.40) hybridized to the catchall and Babesia genus probes but did not hybridize to any species-specific probe tested, suggesting the presence of an unidentified Babesia species. Phylogenetic analysis and sequencing revealed the newly detected Babesia isolate to form a clade distinct from fully identified canine, bovine, equine, and ovine Babesia species, with a range of 88.6-92.9% nucleotide identity.

Stand Transpiration Estimates from Recalibrated Parameters for the Granier Equation in a Chinese Fir (Cunninghamia lanceolata) Plantation in Southern China

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is an important native tree species that is widely distributed in subtropical areas of southern China and cultivated for wood extraction. However, information on water use by Chinese fir plantations is still scarce. In this study, we performed species-specific parameter calibrations for the original Granier equation for sap flow density (SFD) estimates. Stand transpiration (Es) was related to environmental inputs, such as rainfall, air temperature (Ta), vapor pressure deficit (VPD), photosynthetically-active radiation (PAR), air relative humidity (RH), and potential evapotranspiration (PET) in order to examine how environmental factors affect the Es of Chinese fir plantations. According to our results, Granier’s original parameters set underestimated C. lanceolata SFD by up to 54% compared to our species-specific calibrated parameters set. A strong positive correlation was found between SFD, diameter at breast height (DBH), and tree height among trees populations. The total Es in 2015 was 522.1 mm, with obvious seasonal dynamics and an average of 1.4 mm·day−1. Daily and monthly Es were positively correlated with VPD, PAR, Ta, and PET. A negative relationship between Es and RH was detected only at a daily timescale. Our findings indicate that the original Granier equation requires a parameter calibration when it is applied to species-specific thermal dissipation probe (TDP) estimates, and our findings can also provide novel insights on the water use of Chinese fir in major wood production areas in Southern China.

Development of quantitative PCR primers and probes for environmental DNA detection of amphibians in Ontario

DNA from environmental samples (eDNA) is increasingly being used to detect and monitor elusive species. We developed species-specific eDNA primers and probes for qPCR detection of 24 amphibian species native to Ontario, Canada. Cross-species testing confirmed their high specificity and low cross-species amplification, as well as their ability to detect DNA from target species at low concentrations. These detection tools should prove useful for monitoring at-risk amphibian species throughout their native ranges.

Concentrations of environmental DNA (eDNA) reflect spawning salmon abundance at fine spatial and temporal scales

Developing fast, cost-effective assessments of wild animal abundance is an important goal for many researchers, and environmental DNA (eDNA) holds much promise for this purpose. However, the quantitative relationship between species abundance and the amount of DNA present in the environment is likely to vary substantially among taxa and with ecological context. Here, we report a strong quantitative relationship between eDNA concentration and the abundance of spawning sockeye salmon in a small stream in Alaska, USA, where we took temporally- and spatially-replicated samples during the spawning period. This high-resolution dataset suggests that (1) eDNA concentrations vary significantly day-to-day, and likely within hours, in the context of the dynamic biological event of a salmon spawning season; (2) eDNA, as detected by species-specific quantitative PCR probes, seems to be conserved over short distances (tens of meters) in running water, but degrade quickly over larger scales (ca. 1.5 km); and (3) factors other than the mere presence of live, individual fish — such as location within the stream, live/dead ratio, and water temperature — can affect the eDNA-biomass correlation in space or time. A multivariate model incorporating both biotic and abiotic variables accounted for over 75% of the eDNA variance observed, suggesting that where a system is well-characterized, it may be possible to predict species' abundance from eDNA surveys, although we underscore that species- and system-specific variables are likely to limit the generality of any given quantitative model. Nevertheless, these findings provide an important step toward quantitative applications of eDNA in conservation and management.

Searching for a signal: Environmental DNA (eDNA) for the detection of invasive signal crayfish, Pacifastacus leniusculus (Dana, 1852)

Environmental DNA (eDNA) is a rapid, non-invasive method for species detection and distribution using DNA deposited in the environment by target organisms. eDNA has become a recognised and powerful tool for detecting invasive species in a broad range of aquatic ecosystems. We examined the use of eDNA as a tool for detecting the invasive American signal crayfish Pacifastacus leniusculus in Scotland. Species-specific TaqMan probe and primers were designed for P. leniusculus and a robust quantitative PCR (qPCR) assay and DNA extraction protocol were developed. We investigated the detection capability for P. leniusculus from water samples in a controlled laboratory experiment and determined whether crayfish density (low = 1 crayfish 5.5 L-1 or high = 3 crayfish 5.5 L-1) or length of time in tanks (samples taken at 1, 3 and 7 days) influenced DNA detectability. Additionally, the persistence of DNA was investigated after P. leniusculus removal (samples taken at 1, 3 and 7 days post removal). P. leniusculus DNA was consistently detected during the entire 7-day period and higher density tanks yielded stronger positive results with lower Ct values. After removal of P. leniusculus, there was a rapid and continuous decrease in the detectability of DNA. P. leniusculus DNA could only be detected in high density tanks by the end of the 7-day period, while DNA was no longer detectable in low density tanks after 72 hours. Preliminary field experiments sampled water from three sites in winter and five sites in summer. P. leniusculus was known to be present at two of these sites. P. leniusculus was not detected at any site in winter. However, in summer, positive signals were observed at sites with known P. leniusculus, and at sites where P. leniusculus was believed to be present anecdotally, but not confirmed. All sites where crayfish were believed to be absent were negative for eDNA. Therefore, eDNA represents a promising technique to detect and monitor invasive P. leniusculus, although the presence of detectable amounts of eDNA may be season and location dependent, even where invasive crayfish are known to be present.

Identification of pathogenic bacteria in complex samples using a smartphone based fluorescence microscope.

Diagnostics based on fluorescence imaging of biomolecules is typically performed in well-equipped laboratories and is in general not suitable for remote and resource limited settings. Here we demonstrate the development of a compact, lightweight and cost-effective smartphone-based fluorescence microscope, capable of detecting signals from fluorescently labeled bacteria. By optimizing a peptide nucleic acid (PNA) based fluorescence in situ hybridization (FISH) assay, we demonstrate the use of the smartphone-based microscope for rapid identification of pathogenic bacteria. We evaluated the use of both a general nucleic acid stain as well as species-specific PNA probes and demonstrated that the mobile platform can detect bacteria with a sensitivity comparable to that of a conventional fluorescence microscope. The PNA-based FISH assay, in combination with the smartphone-based fluorescence microscope, allowed us to qualitatively analyze pathogenic bacteria in contaminated powdered infant formula (PIF) at initial concentrations prior to cultivation as low as 10 CFU per 30 g of PIF. Importantly, the detection can be done directly on the smartphone screen, without the need for additional image analysis. The assay should be straightforward to adapt for bacterial identification also in clinical samples. The cost-effectiveness, field-portability and simplicity of this platform will create various opportunities for its use in resource limited settings and point-of-care offices, opening up a myriad of additional applications based on other fluorescence-based diagnostic assays.

Molecular Identification of ten species of stored-product psocids through microarray method based on ITS2 rDNA

Stored-product psocids (Psocoptera: Liposcelididae) are cosmopolitan storage pests that can damage stored products and cause serious economic loss. However, because of the body size (~1 mm) of eggs, nymphs, and adults, morphological identification of most stored-product psocids is difficult and hampers effective identification. In this study, 10 economically important stored-product Liposcelis spp. psocids (Liposcelis brunnea, L. entomophila, L. decolor, L. pearmani, L. rufa, L.mendax, L. bostrychophila, L. corrodens, L. paeta, and L. tricolor) were collected from 25 geographic locations in 3 countries (China, Czech Republic, and the United States). Ten species-specific probes for identifying these 10 psocid species were designed based on ITS2 sequences. The microarray method and reaction system were optimized. Specificity of each of the ten probes was tested, and all probes were found suitable for use in identification of the respective10 Liposcelis spp. psocids at 66 °C. This method was also used to identify an unknown psocid species collected in Taian, China. This work has contributed to the development of a molecular identification method for stored-product psocids, and can provide technical support not only to facilitate identification of intercepted samples in relation to plant quarantine, but also for use in insect pest monitoring.

Molecular characterization of interspecific competition of Diachasmimorpha longicaudata (Ashmead) and Fopius arisanus (Sonan) parasitizing the oriental fruit fly, Bactrocera dorsalis (Hendel)

Diachasmimorpha longicaudata (Ashmead) and Fopius arisanus (Sonan), are two of the most abundant and widely used parasitoids for biological control of Bactrocera dorsalis (Hendel) worldwide. However, interspecific competition between them frequently occurs within a single host once they coexit in same orchard, resulting in reduction of their combined impact. However, thus far, it is difficult to characterize these complex interactions. This paper demonstrates qPCR-methods for detection of parasitism for single D. longicaudata, and methods to determine interspecific interaction with F. arisanus. Utilizing qPCR-based methodologies, a species-specific probe was designed for D. longicaudata, which was coupled with a previously published probe for F. arisanus. An assay was developed that is sensitive enough to detect parasitism of solitary D. longicaudata across all developmental stages including one egg per host larva (0.01 ng/40 ng parasitoid/host DNA). The detected parasitism of D. longicaudata was higher than that of using traditional rearing-methods. For the lab-reared and wild strain of multiple species parasitization, qPCR-methods also detected higher parasitism by the above two species from one assay respectively when compared to conventional rearing-based methods. In the later experiment, the majority of D. longicaudata larvae were dying or dead within hollow-like host pupae probably due to interspecific competition. This method is a reliable and robust technique to determine parasitism by a single species or both species of parasitoid across all life stages within one host. It will be useful to evaluate parasitoid combined suppression, and to characterize complex interspecific competition between these two species of parasitoids.

Evaluation of PCR-reverse line blot hybridization assay for simultaneous identification of medically important saprophytic fungi

In immunocompromised patients suffering from invasive fungal infections, rapid identification of fungal species is important since the appropriate treatment is usually related to the responsible species. We describe here, an assay based on combination of PCR and reverse line blot hybridization (PCR/RLB) for differentiation causative agent of fungal infections. We performed PCR/RLB assay on 10 reference strains, which include Aspergillus species (A.fumigatus, A.flavus, A.niger, A.terreus, and A.clavatus), Mucor circnelloides, Rhizopus oryzae, Alternaria alternata, Cladosporium herbarum, and Fusarium solani. Besides, twenty-two clinical specimens from patients with proven fungal infections were analyzed for the identification of species. The obtained results were then compared with the results of culture and sequence analysis. The fungal species-specific oligonucleotide probes were able to distinguish between all species represented in this study with the exception of cross-reactivity between A.niger and A.fumigatus species. Two specimens, which were represented as mixed fungi in culture, were identified properly by this method. Results of the RLB assay were concordant with the culture and ITS sequencing results. Our result demonstrate that the RLB assay potentially is suitable for rapid and simultaneous identification of variety fungal pathogens directly from culture as well as from clinical specimens.

Rapid Detection of Serratia fonticola by TaqMan Quantitative Real-Time PCR Using Primers Targeting the gyrB Gene.

A gyrB gene is present in the majority of bacterial species, and encodes the ATPase domain of DNA gyraseB-subunit protein, which is essential for transcription and replication of bacteria. The gyrB gene exhibits higher nucleotide sequence variability than the 16S rDNA gene and thus could be more reliable in differentiating Serratia fonticola. A species-specific primer pair and probe were designed for quantitative real-time PCR detection of S. fonticola using gyrB as the target gene. Nine members of the Serratia family (representing nine Serratia species) were chosen to verify the specificity of the primers. Additionally, two species each of Salmonella and Klebsiella, and five other species belonging to five other genera of Enterobacteriaceae, were tested for primer cross-reaction. All the tested strains gave negative results. The limit of detection for S. fonticola using the gyrB gene was 100 copies per PCR reaction. This TaqMan PCR assay provided a specific, rapid, and sensitive method to detect S. fonticola based on its gyrB gene.

Identification of Plectropomus leopardus, Promicrops lanceolatus and Cromileptes altivelis using species-specific TaqMan real-time PCR

Plectropomus leopardus, Promicrops lanceolatus and Cromileptes altivelis are high-value groupers in chinese aquatic product market and are substituted by cheaper ones sometimes due to economic profit. In this study, real-time PCR methods for identification of these groupers were developed. Species-specific primers and probes were designed on the basis of cytochrome oxidase I (COI) mitochondrial DNA sequences of groupers and other fishes. The specificity of the real time PCR assays were tested with a total of 51 specimens of fish, meat and poultry. Amplifications were observed in specific species only. The methods developed were accurate and rapid and offered the potential to detect fraudulent or unintentional mislabeling of these species in routine seafood authentication analysis.

Invasive anisakiasis by the parasite Anisakis pegreffii (Nematoda: Anisakidae): diagnosis by real-time PCR hydrolysis probe system and immunoblotting assay

BackgroundAnisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen.MethodsParasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. IgE, IgG4 and IgG immune response against antigens A. pegreffii by Immunoblotting assay was also studied.ResultsAccording to the mtDNA cox2 and the EF1 α − 1 nDNA sequence analysis, the larvae from stomach and intestine of two patients were assigned to A. pegreffii. The Real-time PCR primers/probe system, showed a fluorescent signal at 510 nm for A. pegreffii, in all the three cases.In Immunoblotting assay, patient CC1 showed IgE, IgG4 reactivity against Ani s 13-like and Ani s 7-like; patient CC2 revealed only IgG reactivity against Ani s 13-like and Ani s 7-like; while, the third patient showed IgE and IgG reactivity against Ani s 13-like, Ani s 7-like and Ani s 1-like.ConclusionThe Real-time PCR assay, a more sensitive method than direct DNA sequencing for the accurate and rapid identification of etiological agent of human anisakiasis, was successfully assessed for the first time. The study also highlights the importance to use both molecular and immunological tools in the diagnosis of human anisakiasis, in order to increase our knowledge about the pathological findings and immune response related to the infection by zoonotic species of the genus Anisakis.

Complementary molecular methods detect undeclared species in sausage products at retail markets in Canada

Accurate food labelling is of utmost importance for food safety and consumer choice in the food chain. Complete or partial substitution, whether intentional or unintentional, may introduce food pathogens or allergens to a product or affect personal or religious beliefs. Several studies around the world have reported different degrees of species substitution in meat products but no similar studies have been conducted in the Canadian market for sausage products. In this study, 100 raw meat sausage samples that were labelled as single meat species products (beef, pork, chicken or turkey) were collected from retail establishments across Canada and were surveyed for the presence of a panel of non-labeled species. The predominant meat species were determined using DNA barcoding and contaminant or unclaimed meat species were detected using digital droplet PCR using species specific primers and probes. All samples were also tested for presence of horse meat using real-time PCR. All samples contained the predominant species matching the label species except for five turkey sausage samples which contained chicken as the predominant species. Second, this analysis showed that 6% of beef sausages also contained pork, 20% of chicken sausages contained turkey while 5% contained beef, and 5% of pork sausages also contained beef. Five samples labeled as turkey sausage contained no turkey and one pork sample was found to contain horse meat. The overall mislabeling rate detected in this study was 20% and the results provide a baseline for assessing species mislabeling in processed meat products in Canada.

Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)

Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.), P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum, a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed.The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006ng/μl. Efficiency (E) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E=2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.

A real-time PCR approach to detect predation on anchovy and sardine early life stages

Recruitment of sardine (Sardina pilchardus Walbaum, 1792) and anchovy (Engraulis encrasicolus Linnaeus, 1758) is thought to be regulated by predation of their eggs and larvae. Predators of sardine and anchovy can be identified by visual taxonomic identification of stomach contents, but this method is time consuming, tedious and may underestimate predation, especially in small predators such as fish larvae. Alternatively, genetic tools may offer a more cost-effective and accurate alternative. Here, we have developed a multiplex real-time polymerase chain reaction (RT-PCR) assay based on TaqMan probes to simultaneously detect sardine and anchovy remains in gut contents of potential predators. The assay combines previously described and newly generated species-specific primers and probes for anchovy and sardine detection respectively, and allows the detection of 0,001ng of target DNA (which corresponds to about one hundredth of the total DNA present in a single egg). We applied the method to candidate anchovy and sardine egg predators in the Bay of Biscay, Atlantic Mackerel (Scomber scombrus) larvae. Egg predation observed was limited primarily to those stations where sardine and/or anchovy eggs were present. Our developed assay offers a suitable tool to understand the effects of predation on the survival of anchovy and sardine early life stages.