Identification of Torquetenovirus Species in Patients with Kawasaki Disease Using a Newly Developed Species-Specific PCR Method.

A next-generation sequencing (NGS) study identified a very high viral load of Torquetenovirus (TTV) in KD patients. We aimed to evaluate the feasibility of a newly developed quantitative species-specific TTV-PCR (ssTTV-PCR) method to identify the etiology of KD. We applied ssTTV-PCR to samples collected from 11 KD patients and 22 matched control subjects who participated in our previous prospective study. We used the NGS dataset from the previous study to validate ssTTV-PCR. The TTV loads in whole blood and nasopharyngeal aspirates correlated highly (Spearman's R = 0.8931, p < 0.0001, n = 33), supporting the validity of ssTTV-PCR. The ssTTV-PCR and NGS results were largely consistent. However, inconsistencies occurred when ssTTV-PCR was more sensitive than NGS, when the PCR primer sequences mismatched the viral sequences in the participants, and when the NGS quality score was low. Interpretation of NGS requires complex procedures. ssTTV-PCR is more sensitive than NGS but may fail to detect a fast-evolving TTV species. It would be prudent to update primer sets using NGS data. With this precaution, ssTTV-PCR can be used reliably in a future large-scale etiological study for KD.

Highly specific real-time quantification of diverse microRNAs in human samples using universal primer set frame.

In this study, one group of universal primer set frame, composed by one reverse transcription (RT) primer frame and a pair of quantitative real-time polymerase chain reaction (qRT-PCR) primer frames, was elaborately screened and designed by homebuilt software for sensitive and specific quantification of diverse miRNAs. The universal primer set frame can be applied for multiplex miRNAs detection by simply changing the RT-X part of RT primer frame and RP-Y part of qRT-PCR reverse primer frame based on target sequence. The maximum similarity of RT-Y, RT-Z and qRT-PCR forward primer to the human genome and human transcriptome is less than 76%, ensuring the high specificity in human sample detection. The high sensitivity and broad dynamic linear range of the developed approaches by using designed primer set frame were demonstrated on the in vitro detection of miR-21 and miR-155, with dynamic range of 10 fM to 10 nM and detection limit of 3.74 × 10-15 M and 5.81 × 10-15 M for miR-21 and miR-155, respectively. In particular, the developed assays also have high sequence specificity which could clearly discriminate a single base difference in miRNA sequence. The contents of miR-21 and miR-155 in tissue and serum samples have been successfully detected using the developed assays. Results indicated that miR-21 and miR-155 were elevated in cancer tissue and serum specimens than that of normal samples, implying the developed assays hold a great promise for further application in biomedical research and early clinical diagnosis. More importantly, the primer set frame can be universally used in any miRNA investigation.

Fork PCR: a universal and efficient genome-walking tool.

The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes.

MultiPrime: A reliable and efficient tool for targeted next-generation sequencing.

We present multiPrime, a novel tool that automatically designs minimal primer sets for targeted next-generation sequencing, tailored to specific microbiomes or genes. MultiPrime enhances primer coverage by designing primers with mismatch tolerance and ensures both high compatibility and specificity. We evaluated the performance of multiPrime using a data set of 43,016 sequences from eight viruses. Our results demonstrated that multiPrime outperformed conventional tools, and the primer set designed by multiPrime successfully amplified the target amplicons. Furthermore, we expanded the application of multiPrime to 30 types of viruses and validated the work efficacy of multiPrime-designed primers in 80 clinical specimens. The subsequent sequencing outcomes from these primers indicated a sensitivity of 94% and a specificity of 89%.

An Identification System Targeting the SRK Gene for Selecting S-Haplotypes and Self-Compatible Lines in Cabbage.

Cabbage (Brassica oleracea L. var. capitata) self-incompatibility is important for heterosis. However, the seed production of elite hybrid cannot be facilitated by honey bees due to the cross-incompatibility of the two parents. In this study, the self-compatibility of 58 winter cabbage inbred lines was identified by open-flower self-pollination (OS) and molecular techniques. Based on the NCBI database, a new class I S-haplotype-specific marker, PKC6F/PKC6R, was developed. Verification analyses revealed 9 different S-haplotypes in the 58 cabbage inbred lines; of these lines, 46 and 12 belonged to class I (S6, S7, S12, S14, S33, S45, S51, S68) and class II (S15) S-haplotypes, respectively. The coincidence rate between the self-compatibility index and S-haplotype was 91%. This study developed a Tri-Primer-PCR amplification method to rapidly select plants with specific S-haplotypes in biased segregated S-locus populations. Furthermore, it established an S-haplotype identification system based on these nine S-haplotypes. To overcome parental cross-incompatibility (18-503 and 18-512), an inbred line (18-2169) with the S15 haplotype was selected from the sister lines of self-incompatible 18-512 (S68, class I S-haplotype). The inbred line (18-2169) showed self-compatibility and cross-compatibility with 18-503. This study provides guidance for self-compatibility breeding in cabbage and predicts parental cross-incompatibility in elite combinations.

A manager's guide to using eDNA metabarcoding in marine ecosystems.

Environmental DNA (eDNA) metabarcoding is a powerful tool that can enhance marine ecosystem/biodiversity monitoring programs. Here we outline five important steps managers and researchers should consider when developing eDNA monitoring program: (1) select genes and primers to target taxa; (2) assemble or develop comprehensive barcode reference databases; (3) apply rigorous site occupancy based decontamination pipelines; (4) conduct pilot studies to define spatial and temporal variance of eDNA; and (5) archive samples, extracts, and raw sequence data. We demonstrate the importance of each of these considerations using a case study of eDNA metabarcoding in the Ports of Los Angeles and Long Beach. eDNA metabarcoding approaches detected 94.1% (16/17) of species observed in paired trawl surveys while identifying an additional 55 native fishes, providing more comprehensive biodiversity inventories. Rigorous benchmarking of eDNA metabarcoding results improved ecological interpretation and confidence in species detections while providing archived genetic resources for future analyses. Well designed and validated eDNA metabarcoding approaches are ideally suited for biomonitoring applications that rely on the detection of species, including mapping invasive species fronts and endangered species habitats as well as tracking range shifts in response to climate change. Incorporating these considerations will enhance the utility and efficacy of eDNA metabarcoding for routine biomonitoring applications.

A Comprehensive Evaluation of Enterobacteriaceae Primer Sets for Analysis of Host-Associated Microbiota.

Enterobacteriaceae is one of the most important bacterial groups within the Proteobacteria phylum. This bacterial group includes pathogens, commensal and beneficial populations. Numerous 16S rRNA gene PCR-based assays have been designed to analyze Enterobacteriaceae diversity and relative abundance, and, to the best of our knowledge, 16 primer pairs have been validated, published and used since 2003. Nonetheless, a comprehensive performance analysis of these primer sets has not yet been carried out. This information is of particular importance due to the recent taxonomic restructuration of Enterobacteriaceae into seven bacterial families. To overcome this lack of information, the identified collection of primer pairs (n = 16) was subjected to primer performance analysis using multiple bioinformatics tools. Herein it was revealed that, based on specificity and coverage of the 16S rRNA gene, these 16 primer sets could be divided into different categories: Enterobacterales-, multi-family-, multi-genus- and Enterobacteriaceae-specific primers. These results highlight the impact of taxonomy changes on performance of molecular assays and data interpretation. Moreover, they underline the urgent need to revise and update the molecular tools used for molecular microbial analyses.

A new set of primers for COI amplification from purpleback flying squid (Sthenoteuthis oualaniensis).

Despite the contribution of DNA barcoding towards understanding the biodiversity and distribution of species, the success of the mitochondrial cytochrome c oxidase subunit I gene (COI) amplification has been quite variable when it comes to Cephalopoda. Some species in this class such as Sthenoteuthis oualaniensis seem to be more difficult to amplify COI than others due to failed amplifications with universal primer and lack of specific set of primers. In this study, we developed new Sthenoteuthis - specific primer set, which significantly increased average amplification success. The new primer set will aid the recovery of barcodes from this difficult group and facilitate further studies in phylogeny and cryptic diversity of Sthenoteuthis oualaniensis.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

BACKGROUND: Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. RESULTS: An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. CONCLUSIONS: The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.