Universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR) for SNP genotyping

Abstract Background The detection and identification of single nucleotide polymorphism (SNP) is essential for determining patient disease susceptibility and the delivery of medicines targeted to the individual. At present, SNP genotyping technology includes Sanger sequencing, TaqMan-probe quantitative polymerase chain reaction (qPCR), amplification-refractory mutation system (ARMS)-PCR, and Kompetitive Allele-Specific PCR (KASP). However, these technologies have some disadvantages: the high cost of development and detection, long and time consuming protocols, and high false positive rates. Focusing on these limitations, we proposed a new SNP detection method named universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR). In this method, only two types of fluorescence-labeled probes were used for SNP genotyping, thus greatly reducing the cost of development and detection for SNP genotyping. Results In the amplification process of UPIP-qPCR, unlabeled intermediate primers with template-specific recognition functions could trigger probe hydrolysis and specific signal release. UPIP-qPCR can be used successfully and widely for SNP genotyping. The sensitivity of UPIP-qPCR in SNP genotyping was 0.01 ng, the call rate was more than 99.1%, and the accuracy was more than 99.9%. High-throughput DNA microarrays based on intermediate primers can be used for SNP genotyping. Conclusion This novel approach is both cost effective and highly accurate; it is a reliable SNP genotyping method that would serve the needs of the clinician in the provision of targeted medicine.

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Silica-encapsulated DNA tracers for measuring aerosol distribution dynamics in real-world settings

10.1101/2021.05.19.21257392 ◽

2021 ◽

Author(s):

Anne M Luescher ◽

Julian Koch ◽

Wendelin J Stark ◽

Robert N Grass

Keyword(s):

Real World ◽

Complex Dynamics ◽

Quantitative Polymerase Chain Reaction ◽

Cost Effective ◽

Confined Space ◽

Aerosol Dynamics ◽

Novel Approach ◽

Aerosol Distribution ◽

Wide Range ◽

Series Of Experiments

Aerosolized particles play a significant role in human health and environmental risk management. The global importance of aerosol-related hazards, such as the circulation of pathogens and high levels of air pollutants, have led to a surging demand for suitable surrogate tracers to investigate the complex dynamics of airborne particles in real-world scenarios. In this study, we propose a novel approach using silica particles with encapsulated DNA (SPED) as a tracing agent for measuring aerosol distribution indoors. In a series of experiments with a portable setup, SPED were successfully aerosolized, re-captured and quantified using quantitative polymerase chain reaction (qPCR). Position-dependency and ventilation effects within a confined space could be shown in a quantitative fashion achieving detection limits below 0.1 ng particles per m3 of sampled air. In conclusion, SPED show promise for a flexible, cost-effective and low-impact characterization of aerosol dynamics in a wide range of settings.

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A diagnostic marker kit for Fusarium wilt and sterility mosaic diseases resistance in pigeonpea

Theoretical and Applied Genetics ◽

10.1007/s00122-020-03702-0 ◽

2020 ◽

Author(s):

Rachit K. Saxena ◽

Anil Hake ◽

Abhishek Bohra ◽

Aamir W. Khan ◽

Anupama Hingane ◽

...

Keyword(s):

Fusarium Wilt ◽

Diagnostic Marker ◽

Development Program ◽

Cost Effective ◽

Phenotypic Variance ◽

Breeding Programs ◽

Specific Pcr ◽

Novel Approach ◽

Resistant Varieties ◽

Pathogen Variability

Abstract Fusarium wilt (FW) and sterility mosaic diseases (SMD) are key biotic constraints to pigeonpea production. Occurrence of these two diseases in congenial conditions is reported to cause complete yield loss in susceptible pigeonpea cultivars. Various studies to elucidate genomic architecture of the two traits have revealed significant marker–trait associations for use in breeding programs. However, these DNA markers could not be used effectively in genomics-assisted breeding for developing FW and SMD resistant varieties primarily due to pathogen variability, location or background specificity, lesser phenotypic variance explained by the reported QTL and cost-inefficiency of the genotyping assays. Therefore, in the present study, a novel approach has been used to develop a diagnostic kit for identification of suitable FW and SMD resistant lines. This kit was developed with 10 markers each for FW and SMD resistance. Investigation of the diversity of these loci has shown the role of different alleles in different resistant genotypes. Two genes (C.cajan_03691 and C.cajan_18888) for FW resistance and four genes (C.cajan_07858, C.cajan_20995, C.cajan_21801 and C.cajan_17341) for SMD resistance have been identified. More importantly, we developed a customized and cost-effective Kompetitive allele-specific PCR genotyping assay for the identified genes in order to encourage their downstream applications in pigeonpea breeding programs. The diagnostic marker kit developed here will offer great strength to pigeonpea varietal development program, since the resistance against these two diseases is essentially required for nominating an improved line in varietal release pipeline.

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Novel Dual Labeled Fluorescence Probe Based Assay to Measure the Telomere Length

10.1101/507616 ◽

2018 ◽

Author(s):

Itty Sethi ◽

Gh. Rasool Bhat ◽

Rakesh Kumar ◽

Ekta Rai ◽

Swarkar Sharma

Keyword(s):

Polymerase Chain Reaction ◽

Telomere Length ◽

Fluorescence Probe ◽

Quantitative Polymerase Chain Reaction ◽

Cost Effective ◽

Sybr Green ◽

Telomeric Dna ◽

Chain Reaction ◽

Specific Pcr ◽

Polymerase Chain

ABSTRACTTelomeres are highly repetitive regions capping the chromosomes and composed of multiple units of hexa-nucleotides, TTAGGG, making their quantification difficult. Most of the methods developed to estimate telomeres are extensively cumbersome and expensive. The quantitative polymerase chain reaction (qPCR) based assay is relatively easy and cheaper method that applies SyBr Green dye chemistry to measure telomere length. As SyBr Green dye fluoresces after intercalation into the dsDNA, lack of differentiation between specific PCR target products and unspecific products is a limitation and it affects accuracy in quantitation of telomeres. To overcome the limitations of SyBr Green, we developed a dual labeled fluorescence probe based quantitative polymerase chain reaction (qPCR) to measure the telomere length. This robust, accurate and highly reproducible (R2=0.96) proprietary method (patent pending), yet cost effective and easy, utilizes a probe that targets specifically the telomeric DNA.

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Utilization of DNA microarrays for detection and identification of selected Fusarium species from the Czech Republic

Czech Journal of Food Sciences ◽

10.17221/307/2011-cjfs ◽

2012 ◽

Vol 29 (Special Issue) ◽

pp. S93-S101

Author(s):

L. Pavlátová ◽

D. Novotný ◽

J. Hodek ◽

J. Chrpová ◽

J. Ovesná

Keyword(s):

Dna Microarrays ◽

Fusarium Species ◽

Elongation Factor ◽

Translation Elongation ◽

Specific Pcr ◽

Detection And Identification ◽

Pcr Products ◽

Contaminated Food ◽

Food And Feed ◽

Template Dna

Fusarium is a serious phytopathogenic fungal genus with producting of many kinds of highly toxic secondary metabolites – mycotoxins. The consumption of Fusarium contaminated food and feed can cause dangerous mycotoxicoses both in humans and animals, therefore the detection of a wider range of Fusarium species in the samples of crops is very important. The aim of our work was to test the reliability of detection and identification of three Fusarium species in infected wheat grains by DNA microarrays versus classical mycological methods and by specific PCR. The in-house DNA microarrays for the detection and identification of the selected Fusarium species by using oligonucleotides probes were prepared. For hybridisation on DNA microarrays fluorescent labelled PCR products were used of part of the translation elongation factor 1 alpha. The conditions of hybridisation were optimised on fungal template DNA. The method of DNA microarrays was verified on artificially infected samples of wheat and tested on unknown infected wheat samples with simultaneous analysis by classical mycological methods and by specific PCR.

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Introduction of High Throughput and Cost Effective SNP Genotyping Platforms in Soybean

Plant Genetics Genomics and Biotechnology ◽

10.5147/pggb.v2i1.155 ◽

2017 ◽

Vol 2 (1) ◽

pp. 90-94 ◽

Cited By ~ 2

Author(s):

Jiazheng Yuan ◽

Zixiang Wen ◽

Cuihua Gu ◽

Dechun Wang

Keyword(s):

Cost Effective ◽

Snp Genotyping ◽

Large Sample Size ◽

Specific Pcr ◽

Assay Optimization ◽

Wide Range ◽

Two Systems ◽

Allele Specific ◽

Soybean Plants ◽

Allele Specific Pcr

We presented here the application of two in-plate SNP (single nucleotide polymorphism) genotyping platforms for soybean plants [Glycine max (L.) Merr.], KASP® (Kompetitive Allele Specific PCR genotyping, LGC Genomics) and TaqMan® (Life Technologies) respectively. These two systems offer us an ability to determine the genotypes of 384 individual samples accurately and efficiently by allele specific PCR in a single plate using typical PCR conditions. Both of the systems require small quantity of genomic DNA obtained from a simple DNA extraction. The genomic sequences containing target SNPs can easily be used as a basic blueprint to design the probes and primers of KASP® and TaqMan® assays whether the sequences are obtained from the genome sequence of soybean William 82 (Wm82.a2.v1), Illumina Soy50k SNPs, or parallel resequencing. Moreover, we listed the pros and cons of the two systems and explained the principles behind the platforms. The high call rate and clear clustering separation of the SNPs can be readily obtained from these platforms without conducting any assay optimization processes. These platforms can routinely be performed on 96/384-well plate format with or without an automation procedure. Therefore, these platforms are especially suitable for the SNP genotyping on a particular trait with a large sample size, gene fine mapping, and marker assisted selection. Further, they require little hands-on experience and achieve per-site and per-individual costs below that of current SSR, AFLP, RFLP, and SNP chip technologies. The platforms can be used for genotyping on a wide range of organisms due to their simplicity and flexibility of handling. Meanwhile, we also especially presented some of the advantages using KASP® SNP genotyping pipeline, which was cost effective in the selection of allele specific assay and therefore, efficiently facilitated the soybean genotyping across large numbers (thousands or more) of individual lines for a great range of markers (hundreds to thousands) in our laboratory.

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Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts

Plant Soil and Environment ◽

10.17221/2226-pse ◽

2008 ◽

Vol 53 (No. 3) ◽

pp. 97-104 ◽

Cited By ~ 12

Author(s):

M. Zouhar ◽

M. Marek ◽

O. Douda ◽

J. Mazáková ◽

P. Ryšánek

Keyword(s):

Cost Effective ◽

Healthy Plant ◽

Host Preferences ◽

Sequence Characterized Amplified Region ◽

Detection And Identification ◽

Plant Hosts ◽

Cost Effective Technique ◽

Ditylenchus Dipsaci ◽

Species Specific ◽

Template Dna

Ditylenchus dipsaci, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of D. dipsaci control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of D. dipsaci in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the D. dipsaci stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all D. dipsaci isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect D. dipsaci in artificially infested plant tissues.

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A Novel Approach for the Treatment of Sialolithiasis that Preserves Salivary Duct Anatomy

Annals of Otology Rhinology & Laryngology ◽

10.1177/00034894211018926 ◽

2021 ◽

pp. 000348942110189

Author(s):

Gani Atilla Şengör ◽

Ahmet Mert Bilgili

Keyword(s):

Salivary Gland ◽

Success Rate ◽

Cost Effective ◽

Complete Removal ◽

Level Of Evidence ◽

Invasive Approach ◽

Pneumatic Lithotripsy ◽

Novel Approach ◽

Surgical Methods ◽

Salivary Gland Stones

Objective: The sialendoscopy era in the treatment of salivary gland stones has reduced the use of classical surgical methods. However, the miniature ducts and tools may cause difficulties in removing large sialoliths. Therefore, invasive combined oral surgeries or gland resection may be considered. We searched for the most suitable method in order to stay in line with the minimally invasive approach that preserves the ductus anatomy, and that can reduce the surgical fears of patients. Materials and Methods: The study included 84 cases (23 parotid and 61 submandibular) in whom stones were fragmented by pneumatic lithotripsy and removed between January 2015 and January 2020. The parotid cases comprised 7 females and 16 males, and the submandibular cases comprised 25 females and 36 males. Intraductal lithotripsy was performed using pneumatic lithotripter. This study has fourth level of evidence. Results: Based on total number of cases (n = 84), success rate was 67/84 (79.7%) immediately after sialendoscopy, and overall success rate was 77/84 (91.6%). Based on number of stones treated (n = 111), our immediate success rate was 94/111 (84.6%), and overall success rate was 104/111 (93.7%). The success criteria were complete removal of the stone and fragments in a single sialendoscopy procedure and resolution of symptoms. Conclusions: We successfully treated salivary gland stones, including L3b stones, in our patient cohort with sialendoscopy combined with pneumatic lithotripsy. The lithotripsy method that we have adapted seems to be more useful and cost-effective compared to its alternatives. We were also able to preserve the ductus anatomy and relieve patients’ concerns. Level of Evidence: Level IV

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Using hidden Markov models to find discrete targets in continuous sociophonetic data

Linguistics Vanguard ◽

10.1515/lingvan-2020-0057 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Daniel Duncan

Keyword(s):

Hidden Markov Models ◽

Markov Models ◽

Hidden Markov ◽

Abstract Representation ◽

Language Variation And Change ◽

Novel Approach ◽

Single Target ◽

The Individual ◽

Individual Speaker

Abstract Advances in sociophonetic research resulted in features once sorted into discrete bins now being measured continuously. This has implied a shift in what sociolinguists view as the abstract representation of the sociolinguistic variable. When measured discretely, variation is variation in selection: one variant is selected for production, and factors influencing language variation and change are influencing the frequency at which variants are selected. Measured continuously, variation is variation in execution: speakers have a single target for production, which they approximate with varying success. This paper suggests that both approaches can and should be considered in sociophonetic analysis. To that end, I offer the use of hidden Markov models (HMMs) as a novel approach to find speakers’ multiple targets within continuous data. Using the lot vowel among whites in Greater St. Louis as a case study, I compare 2-state and 1-state HMMs constructed at the individual speaker level. Ten of fifty-two speakers’ production is shown to involve the regular use of distinct fronted and backed variants of the vowel. This finding illustrates HMMs’ capacity to allow us to consider variation as both variant selection and execution, making them a useful tool in the analysis of sociophonetic data.

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Gas Detection and Identification Using Multimodal Artificial Intelligence Based Sensor Fusion

Applied System Innovation ◽

10.3390/asi4010003 ◽

2021 ◽

Vol 4 (1) ◽

pp. 3

Author(s):

Parag Narkhede ◽

Rahee Walambe ◽

Shruti Mandaokar ◽

Pulkit Chandel ◽

Ketan Kotecha ◽

...

Keyword(s):

Sensor Fusion ◽

Network Architecture ◽

Cost Effective ◽

Sensor Data ◽

Dense Layer ◽

Gaseous Emissions ◽

Multiple Sensors ◽

Single Output ◽

Novel Approach ◽

Single Sensor

With the rapid industrialization and technological advancements, innovative engineering technologies which are cost effective, faster and easier to implement are essential. One such area of concern is the rising number of accidents happening due to gas leaks at coal mines, chemical industries, home appliances etc. In this paper we propose a novel approach to detect and identify the gaseous emissions using the multimodal AI fusion techniques. Most of the gases and their fumes are colorless, odorless, and tasteless, thereby challenging our normal human senses. Sensing based on a single sensor may not be accurate, and sensor fusion is essential for robust and reliable detection in several real-world applications. We manually collected 6400 gas samples (1600 samples per class for four classes) using two specific sensors: the 7-semiconductor gas sensors array, and a thermal camera. The early fusion method of multimodal AI, is applied The network architecture consists of a feature extraction module for individual modality, which is then fused using a merged layer followed by a dense layer, which provides a single output for identifying the gas. We obtained the testing accuracy of 96% (for fused model) as opposed to individual model accuracies of 82% (based on Gas Sensor data using LSTM) and 93% (based on thermal images data using CNN model). Results demonstrate that the fusion of multiple sensors and modalities outperforms the outcome of a single sensor.

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HPM: A Hybrid Model for User’s Behavior Prediction Based on N-Gram Parsing and Access Logs

Scientific Programming ◽

10.1155/2020/8897244 ◽

2020 ◽

Vol 2020 ◽

pp. 1-18

Author(s):

Sonia Setia ◽

Verma Jyoti ◽

Neelam Duhan

Keyword(s):

Web Mining ◽

Contextual Information ◽

Hybrid Approach ◽

Web Pages ◽

Continuous Growth ◽

Novel Approach ◽

Content Mining ◽

Long Access ◽

N Gram ◽

The Individual

The continuous growth of the World Wide Web has led to the problem of long access delays. To reduce this delay, prefetching techniques have been used to predict the users’ browsing behavior to fetch the web pages before the user explicitly demands that web page. To make near accurate predictions for users’ search behavior is a complex task faced by researchers for many years. For this, various web mining techniques have been used. However, it is observed that either of the methods has its own set of drawbacks. In this paper, a novel approach has been proposed to make a hybrid prediction model that integrates usage mining and content mining techniques to tackle the individual challenges of both these approaches. The proposed method uses N-gram parsing along with the click count of the queries to capture more contextual information as an effort to improve the prediction of web pages. Evaluation of the proposed hybrid approach has been done by using AOL search logs, which shows a 26% increase in precision of prediction and a 10% increase in hit ratio on average as compared to other mining techniques.

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