17,439 research outputs found
Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits
The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions
Towards Autonomous Microcystin Detection: Investigating Methods for Automation
Due to increased anthropogenic activity, severe eutrophication is occurring in bodies of water around the world. Effects include decreased water quality, decreased value of surrounding land and recreational use (estimated loss in revenue of 0.67 and 3.96 U.S. billion dollars per year), and increased occurrence of toxin producing Harmful Algal Blooms (HABs). Microcystins are cyclic peptides made up of 7 amino acids and 800-1100 Daltons in size. They are one of the most predominantly produced of these toxins, and therefore was the focus of this study. Numerous structural variants of microcystin (referred to as congeners) exist, but microcystin-LR is one of the most common, having a World Health Organization (WHO) recommended limit of 1 µg/L in drinking water. In order to make informed public health decisions on potable and recreational water, an automated in situ instrument for detection of microcystin and its nucleic acids is needed. Very few detection systems have reached the market (i.e. Environmental Sample Processor, McLane Laboratories, USA), but all remain prohibitively costly and complex. Currently, research in many fields is directed towards developing a more cost effective automated in situ detection instrument that can collect and filter environmental samples, extract toxins and nucleic acids, and detect and quantify analytes, genes, and gene transcripts. In this study, a sample preparation method for on-filter collection, filtration, and dual extraction of microcystin and nucleic acids was developed during the summer of 2016 on environmental samples from two bodies of water, Lake Winnebago, WI and Veteran’s Park Lagoon, Milwaukee, WI. Results were compared to a traditional laboratory bead beating method. Results showed that the median extraction ratios (quantified by mass spectrometry) obtained with on-filter method compared to bead beat method (comparative recovery) for microcystin congeners MC-LR, MC-YR, MC-RR, and MC-LA were 43% ± 12%, 34% ± 9%, 46% ± 10% and 44% ± 13%, respectively for Lake Winnebago. The median comparative recovery for MC-LR, MC-YR, and MC-RR was 51% ± 9%, 49% ± 12%, and 53% ± 7%, respectively, for Veteran’s Park Lagoon. Total RNA extraction by the on-filter result showed lower and more inconsistent ratios. Comparative recovery values for the Veteran’s Park Lagoon ranged from 6% to 27% and 5% to 64% for Lake Winnebago. Further quantification with RT-qPCR is needed to evaluate extraction efficiency of the desired gene cluster (mcy). Methods that were evaluated for detection of microcystin included chemical derivatization (fluorescent derivatization) and optical signal amplification (direct and indirect hybridization schemes using DNA aptamers and oligonucleotide probes, nicking enzyme assisted fluorescent signal amplification (NEFSA)). Methods evaluated for detection of nucleic acids included optical signal amplification (direct and indirect hybridization, NEFSA, cascading amplification of nucleic acids (CANA)) and nucleic acid amplification (strand displacement amplification (SDA)). Of the techniques tested, SDA gave non-specific or no amplification, fluorescent derivatization was inconsistent, and all hybridization schemes resulted in non-specific binding. Preliminary results from NEFSA and CANA showed promise, but were inconsistent. Therefore, further optimization of reaction conditions is necessary to conclude if either could be viable options for use in an automated in situ detection system in combination with the on-filter sample preparation and extraction technique
Development And Optimization Of A Multiplex Pcr Assay For Simultaneous Detection Of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Weltevreden, Salmonella Agona, And Salmonella Heidelberg
Salmonella enterica adalah spesis pathogen bawaan makanan penting yang menyebabkan
gastroenteritis dan jangkitan sistemik dalam manusia.
Salmonella enterica are a species of important food-borne pathogens that cause
gastroenteritis and systemic infections in humans. The incidence of food-borne
Salmonellosis due to non-typhoidal Salmonellae (NTS) serotypes is increasing periodically
while typhoidal Salmonellosis (TS) decreases in Malaysia
Introduction to the Synthesis and Purification of Oligonucleotides
Modern nucleic acid synthesizers utilize phosphite triester chemistries that employ stable phosphoramidite monomers to build a growing polymer. These robust reactions allow easy generation of specific oligodeoxyribo‐ and oligoribonucleotides with a variety of labels, modified linkages, and nonstandard bases. Strategies are given for the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143751/1/cpnca03c.pd
The clinical and prognostic use of circulating tumour cells in breast cancer
Adjuvant therapies such as endocrine or cytotoxic chemotherapy have been demonstrated to
improve overall survival in early breast cancer patients. A blood test to monitor patients at risk of
relapse is needed to identify those patients who would benefit from these treatments and those
for whom it is not necessary. This is in favour of detecting disseminated tumour cells (DTCs) from
painful bone marrow aspirates, currently the gold‐standard method for detecting minimal
residual disease (MRD). The use of circulating tumour cells (CTCs) enriched from the blood was
investigated for this purpose along with their characterisation in the metastatic setting to enable
individualised therapy.
Sixty‐four primary breast cancer patients were followed up for up to 12 years post surgery for
any MRD present. This analysis looked at measurements of DTCs in the bone marrow, CTCs in the
blood and circulating‐free DNA (cfDNA) in the plasma over the follow up period. Patients who
had involved lymph nodes at surgery, were significantly more likely to have CTCs present than
low risk patients with no nodes positive, (70% compared to 39% respectively, p = 0.042). Our
analysis also looked at the relationship of cfDNA to DTCs and CTCs. An inverse relationship of cell
death in the blood (manifesting as blood cfDNA) to bone marrow DTCs by qRT‐PCR was apparent.
This may be due to tumour dormancy mechanisms ‐ cycles of tumour cell proliferation and cell
death occurring in the bone marrow, evidence not shown before in patient samples. Combined
use of these markers could therefore be used as a monitoring system for impending metastatic
disease and a rationale for further treatment.
We also participated in a multi–centre study to assess the effects of lapatinib; a targeted therapy
against two members of the human epidermal growth factor receptor family (EGFR and HER2).
This was in advanced breast cancer patients and used CTCs as a surrogate marker. Our study
selected patients on the basis of EGFR positivity in CTCs that were present in the blood. Four out
of 12 patients (33%) demonstrated an initial decrease in the number of EGFR positive CTCs in
response to Lapatinib, however this was limited and all patients were taken off study with
progressive disease. We also explored a novel method in development to detect viable CTCs. This
used an in situ hybridisation method to amplify signals from mRNA transcripts of tumour markers
in CTCs. The use of CTCs is a very useful and promising tool for studying both the biology of
breast cancer, and also as a non‐invasive analytical tool in the clinical setting to gain predictive
and prognostic information
Characterization of Negeviruses and insect virome interactions during co-infection in cell culture
Negeviruses are a newly described taxon of insect-specific viruses (ISVs) that show potential to be used as a virus-based pathogen control strategy through superinfection exclusion. Due to its recent discovery, little is known about the biology of these ISVs or how they interact with the insect’s virome. It was recently demonstrated that both wild-type and genetically modified Negeviruses can inhibit the replication of Alphaviruses in mosquito cell culture. For Negeviruses to be used in wild mosquito populations they will have to compete with other viruses that infect mosquitoes, typically other wild-type Negeviruses and ISVs. Thus, I performed co-infection assays in different cell types to observe homologous and heterologous exclusion during virus infection. The cell lines used were Aag2 cells derived from Aedes aegypti, and C7/10 cells derived from Aedes albopictus. C7/10 cells have a dysfunctional RNA interference response, while Aag2 cells have a functional RNA interference pathway and pre-existing chronic infections with two ISVs: CFAV and PCLV. Homologous exclusion will be tested through additional infectious clones of Negeviruses that have fluorescent reporter genes inserted into their genome. Analyzing the growth trends of different Negeviruses in these cell lines allow us to determine which Negevirus isolates can establish infection in the presence of existing or co-infections with other viruses. Results demonstrated the successful cloning of PIUV ORF3 mScarlet, but with concerns regarding insert stability. The variability in outcomes during co-infection experiments was attributed to cell type differences, mainly RNAi competency, and pre-existing infections. Fitness variations were observed among the viruses, with NEGV isolates and LORV demonstrating higher fitness. These findings contribute to our understanding of Negevirus biology and highlight the importance of further research to overcome the challenges encountered in cloning and characterization of the behavior of these viruses
Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits
The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions
A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer
Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3′ untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries
The Development of a Primer Payload with Microparticles for UTI Pathogen Identification Using Polythymidine- Modified LAMP Primers in Droplet LAMP
Nucleic acid amplification tests (NAATs) are among the diagnostic tests with the highest sensitivity and specificity. However, they are more complex to develop than other diagnostic tests such as biochemical tests and lateral flow immunoassay tests. Polymerase chain reaction (PCR) is the gold standard for NAATs. PCR requires thermal cycling to achieve clonal amplification of the target pathogen DNA for diagnosis. Thermal cycling poses a challenge in the development of PCR diagnostics for point-of-care (POC) settings. Loop-mediated isothermal amplification (LAMP) offers an isothermal method for NAATs diagnostics. The advancement of the microfluidics field significantly enhances the development of LAMP diagnostics devices for POC testing. Another challenge with NAATs, is the limitation in the development of multiplex NAATs. Multiplexing however, occupies an important role in the efforts to address the antimicrobial resistance global crisis. Multiplexing will help to provide more thorough and complete diagnostics of infections, and enable doctors to prescribe the most effective antibiotics to the patients. This will help slow the emergence of antibiotic resistant pathogens. We are currently in a period of discovery void, with regards to antibiotics discovery. At this rate, more pathogens are becoming resistant to the antibiotics that we have, faster than we are developing new classes of antibiotics. According to the World Health Organization (WHO) interagency coordination group on AMR report to the secretary general of the United Nations, by 2050, there will be 10 million annual deaths globally, as a result of AMR-related events. There will also be 1 trillion in healthcare costs, and 28 million people will be living in poverty, as a result of the economic impact of uncontrolled AMR. Another area where multiplex diagnostics play a crucial role is infection control in the era of epidemics and pandemics. The increasing prevailing frequency of global pandemics stresses the need for the development of highly accurate and decentralized POC diagnostics. Over the last ten years, there have been more than 30 epidemics and pandemics around the world, including SARS-CoV-2, Monkey pox, India black fungus, Dengue fever, Measles, Zika, Avian influenza, Influenza A and Ebola. With advancing technology and international commerce and relations, we are now more connected than ever. This means that if there are no developments to make molecular tests more accessible at the POC, the future waves of epidemics and pandemics will have faster spread, further reach and more devastating impacts on the lives of the 8 billion people on our planet. We have developed a diagnostic method for executing droplet microfluidics LAMP via a microparticle primer payload mechanism and have demonstrated it with urinary tract infection (UTI) pathogens. With inspiration from overhang PCR and RNA-Seq, we engineered LAMP primers with 5’ polythymidine (PolyT) oligonucleotide (PolyT is placed in the middle of the Forward inner primers and Backward inner primers). The PolyT sequence is recognized by a biotinylated capture oligonucleotide engineered with a polyadenylated (PolyA) polynucleotide on the 3’ end. The streptavidin-coated microparticles functionalized with the PolyA oligonucleotide and PolyT primers, capture their specific target DNA and deliver the cargo into emulsion droplets of LAMP reagents for amplification. This platform provides the ability to multiplex by coding specific pathogen target DNA with different fluorescent signatures of the microparticles
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