Target DNA Fragments Research Articles

Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system.

The fabrication of nanopores with a matched pore size, and the existence of multiple interferents make the reproducible detection of small-sized molecules by means of solid-state nanopores still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructures related to the existence of small-sized targets. In particular, a DNA tetrahedron with a well-defined 3D nanostructure is the ideal candidate for use as a signal transducer. Here, we demonstrate the detection of an L1-encoding gene of HPV18 as a test DNA target sequence in a reaction buffer solution, where long single-stranded DNA linking DNA tetrahedra onto the surface of the magnetic beads is cleaved by a target DNA-activated CRISPR-cas12 system. The DNA tetrahedra are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) one signal transducer can be used to detect different targets; (2) a glassy nanopore with a pore size much larger than the target DNA fragment can boost the tolerance of the contaminants and interferents which often degrade the performance of a nanopore sensor.

PRIMA: a rapid and cost-effective genotyping method to detect single-nucleotide differences using probe-induced heteroduplexes

Targeted mutagenesis by programmable site-specific nucleases like CRISPR typically produce 1-base pair (bp) insertion or deletion (indel) mutations. Although several methods have been developed to detect such 1-bp indels, each method has pros and cons in terms of cost and/or resolution. Heteroduplex mobility assay (HMA) is a traditional technique detecting small base pair differences but it has a limited resolution of mutation size and the band patterns are often complex. Here, we developed a new method called PRIMA (Probe-Induced HMA) using a short single-stranded DNA molecule as a probe in HMA. By utilizing a 40-mer probe containing a 5-nucleotide deletion, we assessed the mobility of a heteroduplex with a target DNA fragment from a plant, bacterium, and human. This method allowed us to detect a 1-bp indel mutation consistently. We also showed that SNPs can be detected using PRIMA. PRIMA provides a rapid and cost-effective solution for the genotyping.

Latest progress in the study of nanoparticle-based delivery of the CRISPR/Cas9 system.

The CRISPR/Cas9 system has been harnessed to cleave a targeted DNA fragment via its Cas nuclease activity under the direction of guide RNA for rendering gene insertions, deletions, and point mutations in basic research and clinical applications. There are a number of vehicles, including lipofectamine, viruses, and nanoparticles, that can deliver the CRISPR/Cas9 system, but all these methods face numerous challenges during their application in life science contexts. Here, we focus on the delivery of CRISPR/Cas9 via nanoparticles because this method has shown great advantages in terms of safety, simplicity and flexibility.

Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture.

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

Amplification-free detection of bacterial genes using a signaling probe-based DNA microarray

In this study, we developed a novel DNA microarray system that does not require fluorophore-labeling, amplification, or washing of the target nucleic acid fragments. Two types of DNA probes (so-called “signaling probes”) labeled with a fluorescence dye (Cy3) and quencher molecule (BHQ2) were spotted on the DNA microarray such that fluorescent signals of Cy3 could be quenched by BHQ2 due to duplex formation between the probes. The addition of the target DNA or RNA fragments disrupted the duplex formed by the probes, resulting in the generation of fluorescence signals. We examined the assay conditions of the signaling probe-based DNA microarray, including the design of the probes, hybridization temperatures, and methods for fragmentation of target molecules. Since this approach does not require time-consuming processes, including labeling, amplification, and washing, the assay achieved specific detection of 16S rDNA and 16S rRNA extracted from Escherichia coli within 60 min, which was significantly rapid compared to conventional PCR-dependent DNA microarrays.

High-Throughput Identification of Allergens in a Food System via Hybridization Probe Cluster-Targeted Next-Generation Sequencing.

Food allergies (FAs) are a crucial public health problem and a severe food safety issue, resulting in an urgent need for an accurate method to detect all of the hidden allergens that exist in food systems. Current methods for detecting allergens typically utilize ELISA, PCR, or LC-MS, which are suitable for the confirmatory analysis of allergens from ingredients rather than unintended contaminants. In this study, we demonstrate a hybridization probe cluster-targeted next-generation sequencing (HPC-NGS) platform for high-throughput screening of potential allergens in food systems. The HPC-NGS successfully captured target DNA fragments and identified 19 allergenic ingredients in a complex food system. Additionally, the HPC-NGS provided expected allergenic species matching rates of 94.24-100% in single food materials and 99.87-99.98% in processed food products. Thus, HPC-NGS enables the accurate characterization of allergenic ingredients and unintended allergenic contaminants in foods. Our results provide new perspectives on the use of HPC-NGS in the accuracy of high-throughput detection technologies for allergens imposed by the complex matrix effect.

Detection of Small-Sized DNA Fragment in a Glassy Nanopore by Utilization of CRISPR-Cas12a as Converter System

The fabrication of nanopore with matched pore size, and the existence of multi interferents make the reproducible detection of small-sized molecules by means of solid-state nanopore still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructure related to the existence of small-sized target. In particular, DNA tetrahedron with well-defined 3D nanostructure is the ideal candidate to be used as signal transducer. Here, we demonstrate the detection of L1-encoding gene of HPV18 as test DNA target sequence in a reaction buffering solution, where long single-stranded DNA linking DNA tetrahedrons onto the surface of magnetic beads are cleaved by target DNA-activated CRISPR-cas12 system. The DNA tetrahedrons are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) One signal transducer can be used to detect different targets; (2) Glassy nanopore with pore size much larger than the target DNA fragment can boost the tolerance of contaminants and interferents which often degrade the performance of nanopore sensor.

Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters.

Microbial natural products (NPs) are a major source of pharmacological agents. Most NPs are synthesized from specific biosynthetic gene clusters (BGCs). With the rapid increase of sequenced microbial genomes, large numbers of NP BGCs have been discovered, regarded as a treasure trove of novel bioactive compounds. However, many NP BGCs are silent in native hosts under laboratory conditions. In order to explore their therapeutic potential, a main route is to activate these silent NP BGCs in heterologous hosts. To this end, the first step is to accurately and efficiently capture these BGCs. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Herein, we describe recent advances in strategies for BGC cloning, with a focus on the preparation of high-molecular-weight DNA fragment, selection and optimization of vectors used for carrying large-size DNA, and methods for assembling targeted DNA fragment and appropriate vector. The future direction into novel, universal, and high-efficiency methods for cloning NP BGCs is also prospected.

Soil-transmitted helminth infections in free-ranging non-human primates from Cameroon and Gabon

BackgroundZoonotic diseases are a serious threat to both public health and animal conservation. Most non-human primates (NHP) are facing the threat of forest loss and fragmentation and are increasingly living in closer spatial proximity to humans. Humans are infected with soil-transmitted helminths (STH) at a high prevalence, and bidirectional infection with NHP has been observed. The aim of this study was to determine the prevalence, genetic diversity, distribution and presence of co-infections of STH in free-ranging gorillas, chimpanzees and other NHP species, and to determine the potential role of these NHP as reservoir hosts contributing to the environmental sustenance of zoonotic nematode infections in forested areas of Cameroon and Gabon.MethodsA total of 315 faecal samples from six species of NHPs were analysed. We performed PCR amplification, sequencing and maximum likelihood analysis of DNA fragments of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA to detect the presence and determine the genetic diversity of Oesophagostomum spp., Necator spp. and Trichuris spp., and of targeted DNA fragments of the internal transcribed spacer 1 (ITS1) to detect the presence of Ascaris spp.ResultsNecator spp. infections were most common in gorillas (35 of 65 individuals), but also present in chimpanzees (100 of 222 individuals) and in one of four samples from greater spot-nosed monkeys. These clustered with previously described type II and III Necator spp. Gorillas were also the most infected NHP with Oesophagostomum (51/65 individuals), followed by chimpanzees (157/222 individuals), mandrills (8/12 samples) and mangabeys (7/12 samples), with O. stephanostomum being the most prevalent species. Oesophagostomum bifurcum was detected in chimpanzees and a red-capped mangabey, and a non-classified Oesophagostomum species was detected in a mandrill and a red-capped mangabey. In addition, Ternidens deminutus was detected in samples from one chimpanzee and three greater spot-nosed monkeys. A significant relative overabundance of co-infections with Necator and Oesophagostomum was observed in chimpanzees and gorillas. Trichuris sp. was detected at low prevalence in a gorilla, a chimpanzee and a greater spot-nosed monkey. No Ascaris was observed in any of the samples analysed.ConclusionsOur results on STH prevalence and genetic diversity in NHP from Cameroon and Gabon corroborate those obtained from other wild NHP populations in other African countries. Future research should focus on better identifying, at a molecular level, the species of Necator and Oesophagostomum infecting NHP and determining how human populations may be affected by increased proximity resulting from encroachment into sylvatic STH reservoir habitats.Graphical

An efficient marker recycling system for sequential gene deletion in a deep sea-derived fungus Acremonium sp. HDN16-126

Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic, cephalosporin. In light of their significant medical value, an efficient gene disruption method is required for the physiological and biochemical studies on this genus of fungi. However, the number of selection markers that can be used for gene targeting is limited, which constrain the genetic analysis of multiple functional genes. In this study, we established a uridine auxotrophy based marker recycling system which achieves scarless gene deletion, and allows the use of the same selection marker in successive transformations in a deep sea-derived fungus Acremonium sp. HDN16-126. We identified one homologue of Acremonium chrysogenum pyrG (also as a homologous gene of the yeast URA3) from HDN16-126, designated as pyrG-A1, which can be used as a selection marker on uridine free medium. We then removed pyrG-A1 from HDN16-126 genome via homologous recombination (HR) on MM medium with 5-fluoroortic acid (5-FOA), a chemical that can be converted into a toxin of 5-flurouracil by pyrG-A1 activity, thus generating the HDN16-126-△pyrG mutant strain which showed auxotrophy for uridine but insensitivity to 5-FOA and enabled the use of exogenous pyrG gene as both positive and negative selection marker to achieve the scarless deletion of target DNA fragments. We further applied this marker recycling system to successfully disrupt two target genes pepL (encodes a putative 2OG-Fe (II) dioxygenase) and pepM (encodes a putative aldolase) identified from HDN16-126 genome, which are proposed to be functional genes related to 2-aminoisobutyric acid metabolism in fungi. This work is the first application of uridine auxotrophy based scarless gene deletion method in Acremonium species and shows promising potential in assisting sequential genetic analysis of filamentous fungi.

Improving high-resolution copy number variation analysis from next generation sequencing using unique molecular identifiers

BackgroundRecently, copy number variations (CNV) impacting genes involved in oncogenic pathways have attracted an increasing attention to manage disease susceptibility. CNV is one of the most important somatic aberrations in the genome of tumor cells. Oncogene activation and tumor suppressor gene inactivation are often attributed to copy number gain/amplification or deletion, respectively, in many cancer types and stages. Recent advances in next generation sequencing protocols allow for the addition of unique molecular identifiers (UMI) to each read. Each targeted DNA fragment is labeled with a unique random nucleotide sequence added to sequencing primers. UMI are especially useful for CNV detection by making each DNA molecule in a population of reads distinct.ResultsHere, we present molecular Copy Number Alteration (mCNA), a new methodology allowing the detection of copy number changes using UMI. The algorithm is composed of four main steps: the construction of UMI count matrices, the use of control samples to construct a pseudo-reference, the computation of log-ratios, the segmentation and finally the statistical inference of abnormal segmented breaks. We demonstrate the success of mCNA on a dataset of patients suffering from Diffuse Large B-cell Lymphoma and we highlight that mCNA results have a strong correlation with comparative genomic hybridization.ConclusionWe provide mCNA, a new approach for CNV detection, freely available at https://gitlab.com/pierrejulien.viailly/mcna/ under MIT license. mCNA can significantly improve detection accuracy of CNV changes by using UMI.

Electrochemical determination of hantavirus using gold nanoparticle-modified graphene as an electrode material and Cu-based metal-organic framework assisted signal generation

An electrochemical biosensor was prepared for nucleic acid-based hantavirus detection using a Cu-based metal-organic framework (CuMOF) as a signal tag. The CuMOF was synthesized by the solvothermal method and then covalently bonded with signal DNA (sDNA) probes. The Au nanoparticles and reduced graphene oxide composite were deposited on the electrode surface by electroreduction as support substrate and was then functionalized with capture DNA (cDNA) probes by self-assembly. Through the complementary base pairing, the target DNA (tDNA) fragment of hantavirus hybridized with the cDNA and the sDNA in a sandwich-type format. The tDNA was detected according to the current signal of the CuMOF catalyzed reaction using o-phenylenediamine as redox substrate. The peak current of the biosensor at - 0.55V increased linearly in proportion to the logarithmic value of the tDNA concentration from 10-15 to 10-9mol/L, with a detection limit of 0.74 × 10-15mol/L. Moreover, the proposed biosensor was successfully applied to detect hantavirus and was able to distinguish hantavirus from other arboviruses.

Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels.

Primary cardiomyopathies (CMPs) are monogenic but multi-allelic disorders with dozens of genes involved in pathogenesis. The implementation of next-generation sequencing (NGS) approaches has resulted in more time- and cost-efficient DNA diagnostics of cardiomyopathies. However, the diagnostic yield of genetic testing for each subtype of CMP fails to exceed 60%. The aim of this study was to demonstrate that allelic dropout (ADO) is a common phenomenon that reduces the diagnostic yield in primary cardiomyopathy genetic testing based on targeted gene panels assayed on the Ion Torrent platform. We performed mutational screening with three custom targeted gene panels based on sets of oligoprimers designed automatically using AmpliSeq Designer® containing 1049 primer pairs for 37 genes with a total length of 153 kb. DNA samples from 232 patients were screened with at least one of these targeted gene panels. We detected six ADO events in both IonTorrent PGM (three cases) and capillary Sanger sequencing (three cases) data, identifying ADO-causing variants in all cases. All ADO events occurred due to common or rare single nucleotide variants (SNVs) in the oligoprimer binding sites and were detected because of the presence of “marker” SNVs in the target DNA fragment. We ultimately identified that PCR-based NGS involves a risk of ADO that necessitates the use of Sanger sequencing to validate NGS results. We assume that oligoprimer design without ADO data affects the amplification efficiency of up to 0.77% of amplicons.

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study.

The recombination of short homologous ends in Escherichia coli has been known for 30years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

Detection of microamounts of novel coronavirus residues in environment by digital PCR

<p indent="0mm">2019 novel coronaviruses (2019-nCoV) swept the world and are still at a high incidence stage. Highly sensitive and accurate nucleic acid molecular diagnostic techniques and methods have become a hot spot of global concern. 2019-nCoV is a kind of RNA virus. At present, there are many methods to detect virus RNA, including real-time fluorescence quantitative PCR (RT-PCR), colloidal gold detection, chemiluminescence detection, and so on. Colloidal gold detection and chemiluminescence detection need to use monoclonal antibodies against 2019-nCoV, but there are some problems in the preparation of 2019-nCoV monoclonal antibodies, such as long cycle, complex preparation and easy pollution. So the above two methods are not easy to be established for the detection of 2019-nCoV. The superiority of PCR technology is widely used in auxiliary medical clinical diagnosis, customs inspection and quarantine, the development of new agriculture, national defense military defense, and basic research of bioscience, and so on. However, the quantification of the target DNA fragment in RT-PCR is still relatively quantitative. Because this technique mainly depends on CT value and standard curve to quantify the target DNA in the experiment, which affects the amplification efficiency of the whole system, and the sensitivity and accuracy of the reaction system are greatly affected. Because the 2019-nCoV virus is highly contagious, the detection methodology is required to be highly sensitive, which is in line with the characteristics of digital PCR (droplet digital PCR system, referred to as ddPCR). ddPCR has the advantages of absolute quantification, high specificity, high sensitivity and strong interference ability, which can better combine molecular biology and medicine closely, and has a great advantage in the detection of 2019-nCoV. In this study, based on droplet digital PCR detection technology, the detection reagent and method were established using the principle of nested PCR. The Saliva of 333 2019-nCoV patients and their environmental samples were detected and compared with real-time PCR (RT-PCR). The linear range of the droplet digital PCR method established in this study was between 25–5 copies/µL, and minimum detection limit was 0.5 copies/µL, which was 2–3 orders of magnitude higher than that of the commercial real-time fluorescent quantitative PCR detection technology. In 333 samples, 7 were positive in 197 environmental samples, and 9 were positive in 136 human samples. The detection accuracy was 100%, and the environmental micro-virus residues that could not be detected by RT-PCR were detected. The digital PCR detection method established in this study was highly specific, and the minimum detection limit was far lower than that of RT-PCR. It is of great significance for sensitive and accurate detection of 2019-nCoV infection, early infection and environmental microvirus.

CT5, a subtle in vitro DNA assembling method based on the combination of FnCas12a and T5 exonuclease.

To develop a new DNA assembly method based on FnCas12a and T5 exonuclease. We developed a method named as FnCas12a and T5 exonuclease (CT5) cloning system. FnCas12a performs site-directed cleavage to the target DNA fragments, and T5 exonuclease generates 20-30 nt single-stranded region at each end of the DNA fragments for homologous recombination-mediated DNA assembly. CT5 was applied to multi-fragment assembly and DNA cloning of large vectors (> 10kb). The efficiencies were approximately 91.4% and 97%, respectively. In addition, CT5 cloning is also utilized for the "walking" of DNA elements, which enables subtle modification of the relative distances of DNA elements in plasmids. The CT5 method was a precise and exquisite DNA operating system and provided an ideal platform for the study of gene functions, genetic engineering and synthetic biology.

Genome-Wide Identification of Miniature Inverted-Repeat Transposable Elements by Targeted High-Throughput Sequencing.

Miniature inverted-repeat transposable elements (MITEs) are a subset of short, non-autonomous class II transposable elements and also a major source of eukaryotic genomic variation. Therefore, genome-wide identification of MITE insertions can help to shed light on their copy number variation and genome insertion features. Here, we present a protocol for targeted MITE identification and genotyping by high-throughput sequencing. By introducing genome-wide detection of the rice mJing MITE as an example, we describe DNA extraction, DNA fragmentation, targeted DNA fragment enrichment, library construction for high-throughput sequencing, and sequence analysis.

Survey and detection of Banana bunchy top virus in Java

Banana bunchy top virus (BBTV) is one of the most important virus causing disease on banana plantation. Infection of BBTV is indicated by unique symptoms involving yellowing and curling of leaf margin, and stunting of plant. Research was conducted to determine the distribution of banana bunchy top disease (BBTD) in West Java (Bogor and Sukabumi), Central Java (Pati and Magelang), and East Java (Malang and Banyuwangi) based on symptom variations and disease severity. Field samples was then confirmed by polymerase chain reaction (PCR) detection method using four specific primers, each amplified different region of BBTV genome, i.e CP1/2 which amplified coat protein gene in DNA S, mRep1/2, BBT1/2, and BBTV-F/-R which amplified replication gene in DNA R in different region. Symptoms and incidence of BBTD was varied across banana plantation in Java. The most common symptoms observed in the field includes chlorotic, green mosaic, narrow, and upright appearance of the leaves, and stunting of the plant. Incidence of BBTV was found more severe in West Java than those in Central Java and East Java. All primers used in the detection method was successfully amplified the target DNA fragment. DNA R primers were more sensitive than DNA S primers and the better amplification result was achieved using BBT1/2 and BBTVR-F/-R primers.

Novel target capture DNA library preparation method using CircLigase-mediated hook ligation

Target capture DNA library preparation methods primarily use the biotin-streptavidin interaction to enrich target DNA, requiring complex operations and a time-consuming workflow with a series of wash steps at regulated temperatures. Here, a new method is presented termed ‘hook ligation’ for target DNA library preparation, using CircLigase to capture target DNA. The concept was validated by library preparation, sequencing, and acquisition of target DNA fragment information through bioinformatics analysis. An efficient reference point for single-strand DNA ligation to a hook sequence using CircLigase is also provided and it is shown that the efficiency can be influenced by the length and the position of the complementary area on the target DNA.

Compared with conventional PCR assay, qPCR assay greatly improves the detection efficiency of predation.

Studies of predation can contribute greatly to understanding predator–prey relationships and can also provide integral knowledge concerning food webs and multi‐trophic level interactions. Both conventional polymerase chain reaction (cPCR) and quantitative PCR (qPCR) have been employed to detect predation in the field because of their sensitivity and reproducibility. However, to date, few studies have been used to comprehensively demonstrate which method is more sensitive and reproducible in studies of predation. We used a Drosophila melanogaster‐specific DNA fragment (99 bp) to construct a tenfold gradient dilution of standards. Additionally, we obtained DNA samples from Pardosa pseudoannulata individuals that fed on D. melanogaster at various time since feeding. Finally, we compared the sensitivity and reproducibility between cPCR and qPCR assays for detecting DNA samples from feeding trials and standards. The results showed that the cPCR and qPCR assays could detect as few as 1.62 × 103 and 1.62 × 101 copies of the target DNA fragment, respectively. The cPCR assay could detect as few as 48 hr post‐feeding of the target DNA fragment. But the qPCR assay showed that all spiders were positive after consuming prey at various time intervals (0, 24, 48, 72, and 96 hr). A smaller proportion of the technical replicates were positive using cPCR, and some bands on the agarose gel were absent or gray, while some were white and bright for the same DNA samples after amplification by cPCR. By contrast, a larger proportion of the technical replicates were positive using qPCR and the coefficients of variation of the Ct value for the three technical replicates of each DNA sample were less than 5%. These data showed that qPCR was more sensitive and highly reproducible in detecting such degraded DNA from predator's gut. The present study provides an example of the use of cPCR and qPCR to detect the target DNA fragment of prey remains in predator's gut.