scholarly journals Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

2021 ◽  
Author(s):  
Masayuki K. Sakata ◽  
Mone U. Kawata ◽  
Atsushi Kurabayashi ◽  
Takaki Kurita ◽  
Masatoshi Nakamura ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Biodiversity Monitoring ◽  
Natural Ecosystems ◽  
Universal Primer ◽  
Primer Sets

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding-analysis of extra-organismal DNA released into the environment-allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set Amph16S had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

scholarly journals Short COI markers for freshwater macroinvertebrate metabarcoding

2017 ◽  
Author(s):  
Ecaterina Edith Vamos ◽  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Detection Efficiency ◽  
In Silico Analysis ◽  
Environmental Dna ◽  
Degraded Dna ◽  
Macroinvertebrate Communities ◽  
Universal Primer ◽  
Primer Sets ◽  
Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

scholarly journals Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

2020 ◽  
Author(s):  
Florian Leese ◽  
Mandy Sander ◽  
Dominik Buchner ◽  
Vasco Elbrecht ◽  
Peter Haase ◽  
...  
Keyword(s):  
Stream Water ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Reference Database ◽  
Degenerate Primers ◽  
Long Term Ecological Research

AbstractDNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database. However, when universal primers are used on environmental DNA (eDNA) isolated from stream water, macroinvertebrate read and OTU numbers are typically “watered down”, i.e. diluted, compared to whole specimen ‘bulk samples’ due to greater co-amplification of abundant non-target taxa such as algae and bacteria. Because stream macroinvertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3’-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.

scholarly journals Development of a new set of PCR primers for eDNA metabarcoding decapod crustaceans

2019 ◽  
Vol 3 ◽  
Author(s):  
Tomoyuki Komai ◽  
Ryo O. Gotoh ◽  
Tetsuya Sado ◽  
Masaki Miya
Keyword(s):  
Variable Region ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Natural Seawater ◽  
Biodiversity Monitoring ◽  
Decapod Crustaceans ◽  
Important Species ◽  
The Pacific ◽  
Extant Species

The Decapoda is one of the largest orders within the class Malacostraca, comprising approximately 14,000 extant species and including many commercially important species. For biodiversity monitoring in a non-invasive manner, a new set of PCR primers was developed for metabarcoding environmental DNA (eDNA) from decapod crustaceans. The new primers (herein named “MiDeca”) were designed for two conservative regions of the mitochondrial 16S rRNA gene, which amplify a short, hyper-variable region (153–184 bp, 164 bp on average) with sufficient interspecific variations. With the use of MiDeca primers and tissue-derived DNA extracts, we successfully determined those sequences (154–189 bp) from 250 species, placed in 186 genera and 65 families across the suborder Dendrobranchiata and 10 of the 11 infraorders of the suborder Pleocyemata. We also preliminarily attempted eDNA metabarcoding from natural seawater collected at Banda, Tateyama, the Pacific coast of central Japan and detected 42 decapod species including 34 and 8 species with sequence identities of > 98% and 80–98%, respectively. The results suggest the usefulness of eDNA metabarcoding with MiDeca primers for biodiversity monitoring of the decapod species. It appears, however, that further optimisation of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts.

scholarly journals Short COI markers for freshwater macroinvertebrate metabarcoding

2017 ◽  
Author(s):  
Ecaterina Edith Vamos ◽  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Detection Efficiency ◽  
In Silico Analysis ◽  
Environmental Dna ◽  
Degraded Dna ◽  
Macroinvertebrate Communities ◽  
Universal Primer ◽  
Primer Sets ◽  
Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

scholarly journals Quantitative Measure of Small-Subunit rRNA Gene Sequences of the Kingdom Korarchaeota

1998 ◽  
Vol 64 (12) ◽  
pp. 5064-5066 ◽  
Author(s):  
Clifford F. Brunk ◽  
Nicole Eis
Keyword(s):  
Internal Standard ◽  
Pcr Amplification ◽  
Quantitative Measure ◽  
Pcr Primers ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rdna Sequence ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Rdna Sequences

ABSTRACT Comparative PCR amplification of small-subunit (SSU) rRNA gene (rDNA) sequences indicates substantial preferential PCR amplification of pJP27 sequences with korarchaeote-specific PCR primers. The coamplification of a modified SSU rDNA sequence can be used as an internal standard to determine the amount of a specific SSU rDNA sequence.

scholarly journals Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

2019 ◽  
Author(s):  
Elena Valsecchi ◽  
Jonas Bylemans ◽  
Simon J. Goodman ◽  
Roberto Lombardi ◽  
Ian Carr ◽  
...  
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Marine Biodiversity ◽  
Primer Sets ◽  
Marine Megafauna

ABSTRACTMetabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

scholarly journals Failure To Differentiate Cryptosporidium parvum from C. meleagridis Based on PCR Amplification of Eight DNA Sequences

1998 ◽  
Vol 64 (4) ◽  
pp. 1454-1458 ◽  
Author(s):  
Dominique Champliaud ◽  
Philippe Gobet ◽  
Muriel Naciri ◽  
Odile Vagner ◽  
José Lopez ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Dna Sequences ◽  
Target Genes ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
Human Pathogens

ABSTRACT In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of theCryptosporidium genome were evaluated for the detection ofC. parvum, the agent of human cryptosporidiosis, andC. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridiscouldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.

scholarly journals Diversity of Nematode Microbial Antagonists from Algeria Shows Occurrence of Nematotoxic Trichoderma spp.

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 941
Author(s):  
Nawal Benttoumi ◽  
Mariantonietta Colagiero ◽  
Samira Sellami ◽  
Houda Boureghda ◽  
Abdelaziz Keddad ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Fusarium Spp ◽  
Trichoderma Spp ◽  
Bacillus Spp ◽  
Phytoparasitic Nematodes ◽  
Meloidogyne Spp ◽  
Rrna Gene Sequencing

Fungi and bacteria associated to phytoparasitic nematodes Globodera rostochiensis and Meloidogyne spp. in Algeria were identified and characterized. Trichoderma spp. showed the highest prevalence in the cysts of G. rostochiensis. A number of isolates were identified through PCR amplification and the sequencing of the internal transcribed spacer (ITS)1-2 and Rpb2 gene regions. The most represented species were T. harzianum and T. afroharzianum. The latter and T. hirsutum were reported for the first time in Algeria. Fusarium spp., including F. oxysporum and F. solani, comprised a second group of fungi found in cysts. Taxa associated to females of Meloidogyne spp. included T. harzianum, Fusarium spp. and other hyphomycetes. To assess the efficacy of Trichoderma spp., two assays were carried out in vitro with the culture filtrates of two T. afroharzianum and T. harzianum isolates, to check their toxicity versus the second stage juveniles of M. incognita. After 24–48 h exposure, a mortality significantly higher than the control was observed for both filtrates at 1% dilutions. The TRI genes involved in the production of trichothecenes were also amplified with the PCR from some Trichoderma spp. isolates and sequenced, supporting a putative role in nematode toxicity. Bacteria isolated from the cysts of G. rostochiensis included Brucella, Rhizobium, Stenotrophomonas and Bacillus spp., identified through 16S rRNA gene sequencing. The potential of the microbial isolates identified and their mechanisms of action are discussed, as part of a sustainable nematode management strategy.

scholarly journals Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

2000 ◽  
Vol 66 (2) ◽  
pp. 844-849 ◽  
Author(s):  
G. Sabat ◽  
P. Rose ◽  
W. J. Hickey ◽  
J. M. Harkin
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Selective Amplification ◽  
E Coli

ABSTRACT A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminatingE. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl2 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detectingE. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

scholarly journals Detection of Enterotoxic Bacillus cereus andBacillus thuringiensis Strains by PCR Analysis

2001 ◽  
Vol 67 (1) ◽  
pp. 185-189 ◽  
Author(s):  
Bjarne Munk Hansen ◽  
Niels Bohse Hendriksen
Keyword(s):  
Bacillus Cereus ◽  
Protein Complexes ◽  
Pcr Primers ◽  
Gastrointestinal Diseases ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Internal Transcribed Spacer Region ◽  
Primer Sets ◽  
Different Strains

ABSTRACT Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereusand B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceTgene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of thebceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes inB. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

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