scholarly journals In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Manash Jyoti Kalita ◽  
Kalpajit Dutta ◽  
Gautam Hazarika ◽  
Ridip Dutta ◽  
Simanta Kalita ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Sybr Green ◽  
Green Approach ◽  
Novel Approach ◽  
Effective Manner ◽  
Wide Range ◽  
Thermal Cycler

AbstractAs the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

scholarly journals In-house Reverse transcriptase polymerase chain reaction for detection of SARS-CoV2 with increased Sensitivity

2021 ◽  
Author(s):  
Manash Jyoti Kalita ◽  
Kalpajit Dutta ◽  
Gautam Hazarika ◽  
Ridip Dutta ◽  
Simanta Kalita ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Cost Effective ◽  
Sybr Green ◽  
Rdrp Gene ◽  
Green Approach ◽  
Resource Limited ◽  
Effective Manner ◽  
Wide Range ◽  
Primer Sets

Abstract Background:With the increasing COVID-19 infection worldwide, economization of the existing RT-PCR based detection assay becomes the need of the hour. Methods: An assessment of optimal PCR conditions for simultaneous amplification for E, S and RdRp gene of SARS-CoV-2 has been made using both fast traditional and multiplex real time PCR using same primer sets. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19. Results: The designed primers for amplification of E, S and RdRp gene of SARS-Cov-2 in single tube Multiplex PCR amplifications have shown efficient amplification of the target region in 37 minutes using thermal cyclers and 169 minutes with HRM based Real time detection using SYBR green master mix, over a wide range of template concentration, and the results were in good concordance with the commercially available detection kits. Conclusion: This fast HRM based Real time multiplex PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost effective manner apart from the added advantage of primer pair’s compatibility for use in Traditional multiplex PCR, which offers extended applicability of the assay protocol in resource limited setting.

scholarly journals Evaluation of multiplex SYBR green real-time PCR assay for detection of pathogenic Escherichia coli

2020 ◽  
Vol 9 (2) ◽  
pp. 448
Author(s):  
Ema Komalasari ◽  
Winiati P. Rahayu ◽  
Siti Nurjanah
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Rt Pcr ◽  
E Coli ◽  
Melting Curves ◽  
Pathogenic Escherichia Coli ◽  
Wide Range ◽  
Pcr Method

Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.

Identification of New PCR Targets and its Validation for Development of Nucleic Acid-based Detection Assay for Melioidosis

2016 ◽  
Vol 1 (1) ◽  
pp. 18
Author(s):  
Sonia Arora ◽  
Duraipandian Thavaselvam ◽  
Archna Prakash ◽  
Ashu Kumar ◽  
Anita Barua ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Cost Effective ◽  
Sybr Green ◽  
Specific Gene ◽  
Pcr Assay ◽  
Gene Target ◽  
Geographical Regions ◽  
Species Specific

Burkholderia pseudomallei the gram negative, soil saprophyte is the causative agent of melioidosis in human and animals. Development of rapid, sensitive, species specific and cost effective molecular assays are needed for detection of B. pseudomallei from clinical and environmental samples and to differentiate it from other closely related bacterial species. In this study, insilico approach was used to identify new species specific gene targets for molecular diagnosis of B. pseudomallei. The identified targets were then analyzed by SYBR Green real time PCR assay for their specificity, sensitivity and presence across different Indian clinical and soil isolates of B. pseudomallei. Out of the three targets studied SYBR Green real time PCR assay targeting bpss0091 gene of B. pseudomallei was found 100% specific, having detection limit of 12.3fg/µl DNA. The bpss0091 gene target was present in all clinical and soil isolates of B. pseudomallei tested thus suggesting bpss0091 gene based SYBR Green real time PCR assay will be useful for detection of B. pseudomallei in different geographical regions.

scholarly journals Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms

2006 ◽  
Vol 55 (1) ◽  
pp. 43-51 ◽  
Author(s):  
Alex J. Stephens ◽  
Flavia Huygens ◽  
John Inman-Bamber ◽  
Erin P. Price ◽  
Graeme R. Nimmo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Population Structure ◽  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Resolving Power ◽  
Nucleotide Polymorphisms ◽  
Methicillin Resistant ◽  
Effective Manner ◽  
Mrsa Strains

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleotide polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eburst analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbp/sdrE, and the integrated plasmids pT181, pI258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

scholarly journals Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil

2014 ◽  
Vol 11 (1) ◽  
Author(s):  
Alcione de Oliveira dos Santos ◽  
Luan Felipo Botelho Souza ◽  
Lourdes Maria Borzacov ◽  
Juan Miguel Villalobos-Salcedo ◽  
Deusilene Souza Vieira
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Hbv Dna ◽  
Amazon Region ◽  
Wide Range ◽  
Pcr Test

scholarly journals Development of a sensitivity and cost-effective real time pcr: measuring a wide range of HBV DNA concentrations

2013 ◽  
Author(s):  
Alcione Santos ◽  
Luan Souza ◽  
Lourdes Borzacov ◽  
Juan Villalobos-Salcedo ◽  
Deusilene Vieira
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Hbv Dna ◽  
Wide Range

scholarly journals Development of a real-time PCR assay to detect the single nucleotide polymorphism causing Warmblood Fragile Foal Syndrome

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259316
Author(s):  
Sharon Flanagan ◽  
Áine Rowe ◽  
Vivienne Duggan ◽  
Erin Markle ◽  
Maureen O’Brien ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Genetic Mutation ◽  
Wild Type ◽  
High Throughput Analysis ◽  
Single Nucleotide ◽  
Cornell University ◽  
Single Nucleotide Change ◽  
Effective Manner

Warmblood Fragile Foal syndrome (WFFS) is an autosomal recessive condition that affects the maturation of collagen in affected foals. Foals affected with the disease typically die or are euthanised shortly after birth. WFFS is caused by a single nucleotide change at position 2032 of the equine PLOD1 gene, causing an impairment of the wild-type enzyme. A commercial test for the causative genetic mutation is currently available from companies operating under licence from Cornell University but it has limitations. This test requires amplification of a region of the PLOD1 gene encompassing the site of interest, followed by Sanger sequencing of that region and computational analysis. We describe here the development of an alternative, real-time PCR based assay that rapidly and reliably differentiates between the wild-type and WFFS associated nucleotides without the need for sequencing, thus increasing the potential for high throughput analysis of large numbers of samples in a cost-effective manner.

scholarly journals Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles

2018 ◽  
Author(s):  
Joseph Chabi ◽  
Arjen Van’t Hof ◽  
Louis K. N’dri ◽  
Alex Datsomor ◽  
Dora Okyere ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Molecular Diagnostic ◽  
Anopheles Coluzzii ◽  
Wide Range ◽  
Pcr Method ◽  
Dissociation Curves

AbstractThe Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding and molecular diagnostic techniques which are still under improvement even though the current SINE method for identification between An. coluzzii and An. gambiae works reliably. This study describes a refinement of the SINE method to increase sensitivity and high throughput method for the identification of both species and An. arabiensis using amplicon dissociation characteristics.Field collected samples, laboratory reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were provided by the insectary of Vestergaard-NMIMR Vector Labs at the Noguchi Memorial Institute for Medical Research (Ghana) and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye.The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72 Celsius for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.

scholarly journals Introducing Novel Molecular-based Method for Quantification of Homologous Recombination Efficiency

2021 ◽  
Author(s):  
Mustapha Dibbasey ◽  
Terry Gaymes
Keyword(s):  
Homologous Recombination ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Lacz Gene ◽  
Expression Arrays ◽  
Recombinant Product ◽  
Wide Range ◽  
High Level

AbstractBackgroundHomologous recombination (HR) pathway is a DNA double-stranded breaks repair pathway well-known for its high level of accuracy. Low HR pathway efficiency clinically known as homologous recombination deficiency (HRD) was identified in some cancers such as breast and ovarian cancers and studies have reported the sensitivity of HRD cancer cells to DNA repair inhibitors such as Olaparib. However, current techniques including immunofluorescence-based technique are qualitative-based, hence lack sensitivity to determine the functionality of HR pathway. Additionally, some of the techniques including gene expression arrays require expression study of wide range genes involve in HR pathway, which is not cost-effective. The aim of the study is to optimise a PCR-based assay (Norgen’s Homologous Recombination kit) that can be employed to quantitate HR efficiency in cells, which accurately reflects the functional status of HR pathway.Methods and FindingsThe kit has two test plasmids (dl-1 and dl-2) with partial deletions in the LacZ gene and the plasmids are generated from modification of pUC19. HR-proficient (HeLa and AsPC-1) and HR-deficient (CAPAN-1 cells) cancer cell lines were transfected with the two plasmids to generate functional LacZ gene (i.e. recombinant product). The recombinant product was quantified by real-time PCR. Although recombinant product was generated in all the cell lines, our real-time PCR demonstrated a high quantity of recombinant product in HeLa cell line whilst low quantity in CAPAN-1 and AsPC-1 cell lines. The quantity of recombinant product generated and quantified reflects HR pathway efficiency.ConclusionOverall, the results have provided some evidence that the PCR-based kit can be suitably employed for quantification of HR efficiency provided appropriate transfection method and reagent are used. However, further study is required to confirm HR efficiency status of AsPC-1 cells to ascertain the low HR efficiency detected by the kit in these cells.

Multiplex PCR detection of clinical and environmental strains ofVibrio vulnificusin shellfish

2004 ◽  
Vol 50 (11) ◽  
pp. 911-922 ◽  
Author(s):  
Gitika Panicker ◽  
Michael C.L Vickery ◽  
Asim K Bej
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Vulnificus ◽  
High Specificity ◽  
Sybr Green ◽  
Sybr Green I ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
Pcr Method

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 103V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.Key words: Vibrio, multiplex PCR, shellfish, SYBR Green I, real-time PCR.

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