Target Amplicons Research Articles

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

A reference cytochrome c oxidase subunit I database curated for hierarchical classification of arthropod metabarcoding data.

Metabarcoding is a popular application which warrants continued methods optimization. To maximize barcoding inferences, hierarchy-based sequence classification methods are increasingly common. We present methods for the construction and curation of a database designed for hierarchical classification of a 157 bp barcoding region of the arthropod cytochrome c oxidase subunit I (COI) locus. We produced a comprehensive arthropod COI amplicon dataset including annotated arthropod COI sequences and COI sequences extracted from arthropod whole mitochondrion genomes, the latter of which provided the only source of representation for Zoraptera, Callipodida and Holothyrida. The database contains extracted sequences of the target amplicon from all major arthropod clades, including all insect orders, all arthropod classes and Onychophora, Tardigrada and Mollusca outgroups. During curation, we extracted the COI region of interest from approximately 81 percent of the input sequences, corresponding to 73 percent of the genus-level diversity found in the input data. Further, our analysis revealed a high degree of sequence redundancy within the NCBI nucleotide database, with a mean of approximately 11 sequence entries per species in the input data. The curated, low-redundancy database is included in the Metaxa2 sequence classification software (http://microbiology.se/software/metaxa2/). Using this database with the Metaxa2 classifier, we performed a cross-validation analysis to characterize the relationship between the Metaxa2 reliability score, an estimate of classification confidence, and classification error probability. We used this analysis to select a reliability score threshold which minimized error. We then estimated classification sensitivity, false discovery rate and overclassification, the propensity to classify sequences from taxa not represented in the reference database. Our work will help researchers design and evaluate classification databases and conduct metabarcoding on arthropods and alternate taxa.

One-step DNA purification and amplification on an integrated plastic microdevice for on-site identification of foodborne pathogens

In this study, we demonstrate a simple and facile method to enable DNA purification and amplification in a continuous step inside a thermoplastic microdevice. The innate property of thermoplastics was adopted to simplify DNA purification because negatively charged DNA can electrostatically interact with the induced positively charged surfaces of thermoplastics as a sample flows inside a microchannel; thus, the DNA purification step can be eliminated. We report the use of natural plastics for the selective adsorption of DNA and conduct a subsequent flow-through polymerase chain reaction (PCR) using a thermoplastic microdevice. Four thermoplastics—poly(methyl methacrylate) (PMMA), polycarbonates (PC), polystyrene (PS), and polypropylene (PP)—were selected as analytical models, and DNA adsorption phenomena on their pristine surfaces were examined. DNA was successfully immobilized onto the pristine thermoplastic surfaces, and the results were confirmed by fluorescent measurement and contact angle measurement and by performing PCR using a thermocycler. The electrostatically attached DNA was subsequently released from the pristine thermoplastic surface using a PCR reagent, which contained a low ionic strength salt as a component that could be used for DNA elution. The eluted DNA was then seamlessly amplified in the subsequent microchannel designed to perform flow-through PCR. The PCR products were collected by a microchamber connected at the end of the microchannel for on-site fluorescence detection. The PMMA microdevice was used to successfully purify and amplify a 210 bp target amplicon from a foodborne pathogen, Escherichia coli O157:H7, within 35 min. This study is expected to pave the way for developing an integrated monolithic thermoplastic microdevice suitable for rapidly identifying foodborne pathogens with reduced operation, decreased manufacturing cost, and enhanced device disposability.

Target amplicon exome-sequencing identifies promising diagnosis and prognostic markers involved in RTK-RAS and PI3K-AKT signaling as central oncopathways in primary central nervous system lymphoma.

Exome-sequencing for somatic mutation detection and copy number variation analysis are effective and valid methods for evaluating human cancers in current molecular medicine. We conducted target amplicon exome-sequencing analyses using PCR target enrichment and next-generation sequencing on Ion Proton semiconductor sequencers. Twenty-seven primary central nervous system lymphoma (PCNSL) specimens and their corresponding noncancerous tissues were used for multiplex PCR amplification to obtain targeted coverages of the entire coding regions of 409 cancer-related genes. The average of the total numbers of somatic mutations including single-nucleotide variations and insertion/deletion mutations in each specimen was 13.3. Of these, the average of the ratios of nonsynonymous substitutions in each specimen was 74.8%. The most frequent mutations in 27 specimens were in PIM1, MYD88, CD79B, DST, IRF4, ERBB3, MYH11, DCC, and KMT2D. Furthermore, somatic mutations of MYH11 were related to poor prognoses in PCNSL patients. Copy number variations were also duplicated and/or deleted from deep-sequencing in segmental genomic islands. In addition to these prognostic marker candidates, analysis of RTK-RAS-MAPK signaling and the PTEN-PI3K-AKT proapoptotic pathway showed that somatic activations and aberrations, respectively, may be involved in a promising central oncopathway harboring mTOR, c-Myc, FOXO1, and p53. This study provides a foundation for molecular targeted therapies based on genome diagnostics and prognosis in PCNSL.

Microdevice-based solid-phase polymerase chain reaction for rapid detection of pathogenic microorganisms.

We demonstrate the integration of DNA amplification and detection functionalities developed on a lab‐on‐a‐chip microdevice utilizing solid‐phase polymerase chain reaction (SP‐PCR) for point‐of‐need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP‐PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine‐modified forward primers. During SP‐PCR, the immobilized forward primers and freely diffusing fluorescence‐labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP‐PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP‐PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.

Strand Displacement Probes Combined with Isothermal Nucleic Acid Amplification for Instrument-Free Detection from Complex Samples.

Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader’s analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2 750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.

A High-Resolution Digital DNA Melting Platform for Robust Sequence Profiling and Enhanced Genotype Discrimination.

DNA melting analysis provides a rapid method for genotyping a target amplicon directly after PCR amplification. To transform melt genotyping into a broad-based profiling approach for heterogeneous samples, we previously proposed the integration of universal PCR and melt analysis with digital PCR. Here, we advanced this concept by developing a high-resolution digital melt platform with precise thermal control to accomplish reliable, high-throughput heat ramping of microfluidic chip digital PCR reactions. Using synthetic DNA oligos with defined melting temperatures, we characterized sources of melting variability and minimized run-to-run variations. Within-run comparisons throughout a 20,000-reaction chip revealed that high-melting-temperature sequences were significantly less prone to melt variation. Further optimization using bacterial 16S amplicons revealed a strong dependence of the number of melting transitions on the heating rate during curve generation. These studies show that reliable high-resolution melt curve genotyping can be achieved in digital, picoliter-scale reactions and demonstrate that rate-dependent melt signatures may be useful for enhancing automated melt genotyping.

Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. The major disadvantage of LAMP is its heavy reliance on indirect detection methods like turbidity and non-specific dyes, which often leads to the detection of false positive results. In the present work, we have developed a direct detection approach, whereby a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. The FLOS-LAMP had a limit of detection of 500 copies of the target with a clinical sensitivity and specificity of 96.8% and 100%, respectively. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. false positives. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.

Apical periodontitis: preliminary assessment of microbiota by 16S rRNA high throughput amplicon target sequencing

BackgroundApical periodontitis includes periapical granulomas and radicular cysts, which are histologically distinguished by the absence and the presence of an epithelial lining, respectively. The main cause of apical periodontitis is the bacterial colonization of the root canal space. This research aimed at assessing whether and how periapical granulomas and radicular cysts differ in terms of microbiota using high throughput amplicon target sequencing (HTS) techniques.MethodsThis study included 5 cases of Periapical Granulomas (PGs) and 5 cases of Radicular Cysts (RCs) selected on the base of histology out of 37 patients from January 2015 to February 2016. Complete medical history, panoramic radiograms (OPTs) and histologic records of each patient were assessed. Only lesions greater than 1 cm in diameter and developed in proximity to teeth with bad prognosis were included. The microbiota present in periapical granulomas and radicular cysts thus retrieved was finely characterized by pyrosequencing of the 16S rRNA genes.ResultsThe core of OTUs shared between periapical granulomas and radicular cysts was dominated by the presence of facultative anaerobes taxa such as: Lactococcus lactis, Propionibacterium acnes, Staphylococcus warneri, Acinetobacter johnsonii and Gemellales. L. lactis, the main OTUs of the entire datasets, was associated with periapical granuloma samples. Consistently with literature, the anaerobic taxa detected were most abundant in radicular cyst samples. Indeed, a higher abundance of presumptive predicted metabolic pathways related to Lipopolysaccharide biosynthesis was found in radicular cyst samples.ConclusionsThe present pilot study confirmed the different microbial characterization of the two main apical periodontitis types and shade light on the possible role of L. lactis in periapical granulomas.

Prevalence of salivary human herpesviruses in pediatric multiple sclerosis cases and controls

Background: Multiple sclerosis (MS) is a multifactorial disease of unknown origin. The current paradigm is that disease develops in genetically susceptible individuals, influenced by environmental factors. Epstein–Barr virus (EBV) and human herpesvirus 6 (HHV-6) have particularly strong associations with the disease. Both viruses are typically acquired during childhood, decades before MS presents. However, in patients with pediatric MS, the temporal window between viral acquisition and disease onset is shortened, which may provide insights into the association of herpesviruses with MS. Objective: To compare the frequency of EBV and HHV-6 in the saliva of a cohort of pediatric MS patients and age-matched controls. Methods: The study enrolled 32 pediatric MS patients and 42 controls and evaluated saliva for HHV-6 u57 and EBV lmp-1 amplification by droplet digital polymerase chain reaction (ddPCR). Results: Pediatric MS patients did not differ from controls in the frequency or magnitude of salivary viral shedding. During the assessment of EBV positivity, distinct profiles emerged that correlated with target amplicon mutations. Conclusions: None of these mutations were evident in EBV-positive samples from pediatric MS patients, whereas they were present in pediatric controls, in addition to MS and control adults, suggesting differential host-immune control of EBV in this pediatric MS cohort.

Double gene targeting PCR assay for the detection of Crocodylus porosus in commercial products

ABSTRACTThe demand for crocodile meat is quickly growing because of its exotic and organoleptic appeal and also the low content of cholesterol and lipids. Moreover, crocodile oil and blood have been used in alternative medicines for treating asthma and several other ailments since ancient times. Furthermore, crocodile hides have great demand in leather industries. All of these have collectively contributed to the extensive hunting, illegal trading and consequent decline of crocodiles in most parts of the world. To keep space with the growing demands, some crocodile species such as Crocodylus porosus have been raised in farms and its commercial trades have been legalised. However, demand for wild crocodiles in foods and medicines has continued in high gear. Recently, several DNA-based methods have been proposed for crocodile detection, but those assays are based on single gene and longer-sized amplicon targets that break down during extensive processing. To address this gap, here we developed and validated a highly stable double gene targeted multiplex PCR assay for the identification of C. porosus materials in commercial products. The assay involved two short sites from C. porosus atp6 (77 bp) and cytb (127 bp) genes and a universal internal control (99 bp) for eukaryotes. The PCR primers were cross-tested against 18 species and validated under pure and mixed matrices under extensive boiling, autoclaving and microwave cooking conditions. Finally, it was used to identify five crocodile-based commercial products. The lower limits of detection for atp6 and cytb genes were 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% under mixed matrices. Some inherent features, such as 77–127 bp amplicon sizes, exceptional stability and superior sensitivity, suggested the assay could be used for the identification of C. porosus in any forensic specimen.

Identification of somatic mutations in postmortem human brains by whole genome sequencing and their implications for psychiatric disorders.

Somatic mutations in the human brain are hypothesized to contribute to the functional diversity of brain cells as well as the pathophysiology of neuropsychiatric diseases. However, there are still few reports on somatic mutations in non-neoplastic human brain tissues. This study attempted to unveil the landscape of somatic mutations in the human brain. We explored the landscape of somatic mutations in human brain tissues derived from three individuals with no neuropsychiatric diseases by whole-genome deep sequencing at a depth of around 100. The candidate mutations underwent multi-layered filtering, and were validated by ultra-deep target amplicon sequencing at a depth of around 200 000. Thirty-one somatic mutations were identified in the human brain, demonstrating the utility of whole-genome sequencing of bulk brain tissue. The mutations were enriched in neuron-expressed genes, and two-thirds of the identified somatic single nucleotide variants in the brain tissues were cytosine-to-thymine transitions, half of which were in CpG dinucleotides. Our developed filtering and validation approaches will be useful to identify somatic mutations in the human brain. The vulnerability of neuron-expressed genes to mutational events suggests their potential relevance to neuropsychiatric diseases.

Development of genic-SSR markers and genetic diversity of Indian lettuce (Lactuca indica L.) in South Korea.

Indian lettuce (Lactuca indica L.) is an undomesticated wild plant with high economic potential. We know little about the plant's genome, such as its DNA markers, making genetic research using this plant difficult. In this study, 100 genic simple sequence repeat (SSR) primers with a 99-250bp target amplicon were synthesized from L. indica transcriptomic sequences. These primers were examined in 8 diverse L. indica accessions, and 90 polymorphic SSRs were obtained. Twenty-three of the 90 polymorphic SSRs were used to investigate transferability to another two Lactuca species, Lactuca serriola and Lactuca sativa. Genetic diversity was investigated in 77 Lactuca accessions, including 73 L. indica collected from across South Korea, 2 L. serriola, and 2 L. sativa. Our genic-SSR markers were highly polymorphic with a mean polymorphic information content of 0.61 and, on average, 10.83 alleles per locus. The average expected heterozygosity (0.76) was higher than the observed heterozygosity. An analysis of molecular variance revealed that most of the total variance in our population is attributable to genetic variation among accessions, rather than among provinces. STRUCTURE, unweighted neighbor-joining phylogenetic trees, and principal coordinate analyses resulted in three clusters, where northern and central-southern L. indica accessions were grouped into two clusters with some admixture. The L. serriola and L. sativa accessions did not produce a separate cluster due to a small sample size. These results show our SSR markers will be useful in germplasm assessment and genetic studies of L. indica and other Lactuca species.

MiSeq: A Next Generation Sequencing Platform for Genomic Analysis.

MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing solutions. The MiSeq instrument is one of the smallest benchtop sequencers that can perform onboard cluster generation, amplification, genomic DNA sequencing, and data analysis, including base calling, alignment and variant calling, in a single run. It performs both single- and paired-end runs with adjustable read lengths from 1×36 base pairs to 2×300 base pairs. A single run can produce output data of up to 15Gb in as little as 4h of runtime and can output up to 25M single reads and 50M paired-end reads. Thus, MiSeq provides an ideal platform for rapid turnaround time. MiSeq is also a cost-effective tool for various analyses focused on targeted gene sequencing (amplicon sequencing and target enrichment), metagenomics, and gene expression studies. For these reasons, MiSeq has become one of the most widely used next generation sequencing platforms. Here, we provide a protocol to prepare libraries for sequencing using the MiSeq instrument and basic guidelines for analysis of output data from the MiSeq sequencing run.

Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

Targeted Next-Generation Sequencing Identification of Mutations in Disease Resistance Gene Analogs (RGAs) in Wild and Cultivated Beets

Resistance gene analogs (RGAs) were searched bioinformatically in the sugar beet (Beta vulgaris L.) genome as potential candidates for improving resistance against different diseases. In the present study, Ion Torrent sequencing technology was used to identify mutations in 21 RGAs. The DNA samples of ninety-six individuals from six sea beets (Beta vulgaris L. subsp. maritima) and six sugar beet pollinators (eight individuals each) were used for the discovery of single-nucleotide polymorphisms (SNPs). Target amplicons of about 200 bp in length were designed with the Ion AmpliSeq Designer system in order to cover the DNA sequences of the RGAs. The number of SNPs ranged from 0 in four individuals to 278 in the pollinator R740 (which is resistant to rhizomania infection). Among different groups of beets, cytoplasmic male sterile lines had the highest number of SNPs (132) whereas the lowest number of SNPs belonged to O-types (95). The principal coordinates analysis (PCoA) showed that the polymorphisms inside the gene Bv8_184910_pkon (including the CCCTCC sequence) can effectively differentiate wild from cultivated beets, pointing at a possible mutation associated to rhizomania resistance that originated directly from cultivated beets. This is unlike other resistance sources that are introgressed from wild beets. This gene belongs to the receptor-like kinase (RLK) class of RGAs, and is associated to a hypothetical protein. In conclusion, this first report of using Ion Torrent sequencing technology in beet germplasm suggests that the identified sequence CCCTCC can be used in marker-assisted programs to differentiate wild from domestic beets and to identify other unknown disease resistance genes in beet.

Dengue IgG Seropositivity and Zika Viral Load

BackgroundSecondary dengue virus (DENV) infections are typically more severe than primary infections. It is not known whether previous DENV infection is associated with higher Zika virus (ZIKV) quantitative RT-PCR results (viral loads (VLs)) in areas endemic for DENV such as Puerto Rico. Our objective was to analyze the association between previous DENV infection (DENV IgG-positive) and ZIKV VL among children with symptomatic ZIKV infection enrolled in the Sentinel Enhanced Dengue and Acute Febrile Illness Surveillance System (SEDSS) in Puerto Rico.MethodsThe study population for this analysis comprised individuals <18 years of age enrolled in SEDSS during 2016 who were ZIKV PCR-positive in serum (using the CDC Trioplex RT-polymerase chain reaction (RT-PCR) assay) within 5 days post-onset (DPO) of symptoms. ZIKV VLs (genome copies/mL) were determined using an RNA standard curve generated from the RT-PCR assay target amplicons. An in-house ELISA was used to ascertain the presence or absence of serum DENV IgG. Trends were assessed using Jonckheere-Terpstra and Chi-square for proportions tests. The Mann–Whitney-Wilcoxon test was used to compare medians. Linear regression modeling was used to determine the association between DENV IgG and ZIKV VL.ResultsOf the 319 individuals who met inclusion criteria, 163 have dengue IgG assays completed to date. Of these, 90/163 (55%) were DENV IgG-positive and 73/163 (45%) were DENV IgG-negative, and did not vary by sex (P = 1.00). However, the proportion of patients with DENV IgG-positivity increased with age (P < 0.001) (Figure). Overall, the median (interquartile range, IQR) ZIKV VL was 23,110 (7,452–84,003), and did not vary by age (P = 0.11) or sex (P = 0.33). However, the median ZIKV VL varied by DPO: 26,230 (DPO<3; n = 117), 15,159 (DPO≥ 3; n = 46), P = 0.002. The median (IQR) ZIKV VLs were: 24,073 (10,938–73,130) in DENV IgG-negative specimens and 22,658 (7,332–89,322) in DENV IgG-positive specimens (P = 0.91). Linear regression indicated no association between DENV IgG and ZIKV VL (P = 0.54).ConclusionDENV IgG-positivity increased with age among children with symptomatic ZIKV infection. ZIKV VLs did not vary by age, but decreased with increasing DPO. There was no association between DENV IgG and ZIKV VL.Disclosures All authors: No reported disclosures.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P.damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P.damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity

Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies.IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.