The web-based multiplex PCR primer design software Ultiplex and the associated experimental workflow: up to 100- plex multiplicity

Background A large number of variants have been employed in various medical applications, such as providing medication instructions, disease susceptibility testing, paternity testing, and tumour diagnosis. A high multiplicity PCR will outperform other technologies because of its lower cost, reaction time and sample consumption. To conduct a multiplex PCR with higher than 100 plex multiplicity, primers need to be carefully designed to avoid the formation of secondary structures and nonspecific amplification between primers, templates and products. Thus, a user-friendly, highly automated and highly user-defined web-based multiplex PCR primer design software is needed to minimize the work of primer design and experimental verification. Results Ultiplex was developed as a free online multiplex primer design tool with a user-friendly web-based interface (http://ultiplex.igenebook.cn). To evaluate the performance of Ultiplex, 294 out of 295 (99.7%) target primers were successfully designed. A total of 275 targets produced qualified primers after primer filtration, and 271 of those targets were successfully clustered into one compatible PCR group and could be covered by 108 primers. The designed primer group stably detected the rs28934573(C > T) mutation at lower than a 0.25% mutation rate in a series of samples with different ratios of HCT-15 and HaCaT cell line DNA. Conclusion Ultiplex is a web-based multiplex PCR primer tool that has several functions, including batch design and compatibility checking for the exclusion of mutual secondary structures and mutual false alignments across the whole genome. It offers flexible arguments for users to define their own references, primer Tm values, product lengths, plex numbers and tag oligos. With its user-friendly reports and web-based interface, Ultiplex will provide assistance for biological applications and research involving genomic variants.

Development of a LAMP-Based Molecular Species Diagnosis Method for Four Major Agricultural Pests in the Genus Spodoptera (Lepidoptera: Noctuidae)

Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major Spodoptera pests: S. exigua, S. frugiperda, S. litura, and S. littoralis. S. exigua and S. litura mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for S. exigua, and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng–1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.

Development of a Nested-MultiLocus Sequence Typing approach for a highly sensitive and specific identification of Xylella fastidiosa subspecies directly from plant samples

Different sequence types (ST) of Xylella fastidiosa were already identified in France and Spain based on direct MultiLocus Sequence Typing (MLST) of plant DNA samples. However, direct typing of plant DNA is partly efficient. In order to improve the sensitivity of X. fastidiosa identification, we developed a direct nested-MLST assay on plant extracted DNA. This method was performed based on a largely used scheme targeting seven housekeeping gene (HKG) loci (cysG, gltT, holC, leuA, malF, nuoL, petC). Nested primers were designed from multi-sequence alignments of 38 genomes representing all subspecies and one genome of Xylella taiwanensis. Sequences obtained were long enough to be used for BLAST comparison in PubMLST database. No nonspecific amplification products were observed in these samples. Efficiency of the nested-MLST was tested on extracted DNA from 106 samples proven positive (Cq<35) or equivocal (35≤Cq≤40) using the Harper’s qPCR test. Samples analyzed included 49 plant species and two insect species (Philaenus spumarius, Neophilaenus campestris) that were collected in 2017 (106 plant samples in France), in 2018 (162 plant samples in France, 40 plant samples and 26 insect samples in Spain), and in 2019 (30 plant samples in Spain). With the conventional-MLST assay, no complete MLST profile was obtained for any of the samples from France and for most samples (59/66) from Spain. Conversely, with the nested approach, complete profiles were obtained for six French plant samples, 55 Spanish plant samples and nine Spanish insect samples. The threshold was improved by 100 to 1000 times compared to conventional PCR and was between 22 pg.mL−1 to 2.2 pg.mL−1 depending on the HKG. Using nested-MLST assay, plants that were not yet considered hosts tested positive and revealed novel alleles in France, whereas for Spanish samples it was possible to assign the subspecies or ST to samples considered as new hosts in Europe. Direct typing by nested-MLST from plant material has an increased sensitivity and may be useful for epidemiological purposes.

Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum

ABSTRACT Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Development and Characterization of Simple Sequence Repeat Markers for, and Genetic Diversity Analysis of Liquidambar formosana

Liquidambar formosana (Hamamelidaceae) is a relatively fast-growing deciduous tree of high ornamental value that is indigenous to China. However, few molecular markers are available for the species or its close relatives; this has hindered genomic and genetic studies. Here, we develop a series of transferable expressed sequence tag-simple sequence repeats (EST-SSRs) for genomic analysis of L. formosana. We downloaded the sequence of the L. formosana transcriptome from the National Center of Biotechnology Information Database and identified SSR loci in the Unigene library. We found 3284 EST-SSRs by mining 34,491 assembled unigenes. We synthesized 100 random primer pairs for validation of eight L. formosana individuals; of the 100 pairs, 32 were polymorphic. We successfully transferred 12 EST-SSR markers across three related Liquidambar species; the markers exhibited excellent cross-species transferability and will facilitate genetic studies and breeding of Liquidambar. A total of 72 clones of three Liquidambar species were uniquely divided into three main clusters; principal coordinate analysis (PCoA) supported this division. Additionally, a set of 20 SSR markers that did not exhibit nonspecific amplification were used to genotype more than 53 L. formosana trees. The mean number of alleles (Na) was 5.75 and the average polymorphism information content (PIC) was 0.578, which was higher than that of the natural L. formosana population (0.390). In other words, the genetic diversity of the plus L. formosana population increased, but excellent phenotypic features were maintained. The primers will be valuable for genomic mapping, germplasm characterization, gene tagging, and further genetic studies. Analyses of genetic diversity in L. formosana will provide a basis for efficient application of genetic materials and rational management of L. formosana breeding programs.

CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification

False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.

Isothermal digital detection of microRNAs using background-free molecular circuit

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.

Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts

ABSTRACT Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms) or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucial for evaluating a marker gene’s true- or false-positive reactions. Here, we analyzed V6 region 16S rRNA gene sequences from 257 animal fecal samples and tested a subset of 184 using qPCR for human/sewage marker genes. Overall, specificities of human/sewage marker genes within sequencing data were 99.6% (BacV6-21), 96.9% (Lachno3), and 96.1% (HF183, indexed by its inferred V6 sequence). Occurrence of some true cross-reactions was associated with atypical compositions of organisms within the genera Blautia or Bacteroides. For human/sewage marker qPCR assays, specificities were 96.7% (HF183/Bac287R), 96.2% (BacV6-21), 95.6% (human Bacteroides [HB]), and 94.0% (Lachno3). Select assays duplexed with either Escherichia coli or Enterococcus spp. were also validated. Most of the positive qPCR results in animals were low level and, on average, 2 orders of magnitude lower than the copy numbers of E. coli and Enterococcus spp. The lower specificity in qPCR assays compared to sequencing data was mainly caused by amplification of sequences highly similar to the marker gene and not the occurrence of the exact marker sequence in animal fecal samples. IMPORTANCE Identifying human sources of fecal pollution is critical to remediate sanitation concerns. Large financial investments are required to address these concerns; therefore, a high level of confidence in testing results is needed. Human fecal marker genes validated in this study showed high specificity in both sequencing data and qPCR results. Human marker sequences were rarely found in individual animals, and in most cases, the animals had atypical microbial communities. Sequencing also revealed the presence of closely related organisms that could account for nonspecific amplification in certain assays. Both the true cross-reactions and the nonspecific amplification had low signals well below E. coli or Enterococcus levels and likely would not impact the assay’s ability to reliably detect human fecal pollution. No animal source had multiple human/sewage marker genes present; therefore, using a combination of marker genes would increase the confidence of human fecal pollution detection.

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection

Background: Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is qRT-PCR, yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito RNA. In this work we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing for confident detection.Methods: Our new oligo design to detect ZIKV RNA included in silicoanalysis of both viral and mosquito (Ae. aegyptiand Ae. albopictus)genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedesgenomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes.Standard curves constructed with in vitrotranscribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay.Results: Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegyptiimmune response to virus infection.Conclusions: The experimental approach to detect ZIKV RNA in Aedes aegyptipresented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

A Novel Isothermal Amplification Method for Detecting Mouse Source Component in Meat

Background: The problem of adulterated meat has become one of the greatest food safety issues in the world. It is reported that the meat used for adulteration includes fox meat, raccoon meat, mink meat, mouse meat, and so on. Although this kind of meat is edible in some areas, such meat is potentially harmful to human health because it is easy to carry bacteria, viruses, and harmful substances. The harm of mouse meat is most frightening. Therefore, it is urgent to develop a fast, accurate, and simple method to effectively identify mouse meat. Methods: In the present study, a new method of isothermal amplification based on the 16S ribosomal RNA gene of the mitochondrial DNA of the mouse was developed. The method is meant to improve the loop-mediated isothermal amplification (LAMP), separating the forward inner primers and backward inner primers, greatly reducing the nonspecific amplification of the method. Results: We have successfully obtained a set of best primers. The developed system allowed for the detection of 0.5% mouse meat from meat mixture effectively and specifically. The best ratio of the primers (F3: F2: F1: RF) was 1:2:2:8, and the optimum concentration of DNA template was 0.35 ng/μL. Conclusions: The assay has great specificity and sensitivity for detecting mouse meat and could provide specific positive results within 1 h. Highlights: We found a new approach of isothermal amplification to detect mouse source components. The LOD is determined to be 0.5 mg/mg. This new method is easy to perform and is able to provide rapid results in the specific detection of mouse meat sources.