Real-time PCR Thermocycler Research Articles

Evaluating sensitivity and specificity of the Biomeme Franklin™ three9 real-time PCR device and SARS-CoV-2 go-strips assay using clinical samples.

We evaluated the sensitivity and specificity of the Biomeme Franklin™ three9 Real-Time PCR Thermocycler and Biomeme SARS-CoV-2 Go-Strips in the detection of SARS-CoV-2. The Biomeme Franklin™ three9 platform is a portable, battery-operated system that could be used in remote settings. We assessed performance of the Biomeme SARS-CoV-2 detection system at a wide range of viral concentrations, examined cross-reactivity of the SARS-CoV-2 Go-Strips against several near-neighbor respiratory pathogens, and evaluated agreement against the BioFire® Respiratory Panel 2.1 in four clinical sample types. Our data indicate the Biomeme Go-Strips can reliably detect SARS-CoV-2 at a concentration of 4.2 × 103 copies/mL. No cross reactivity of the Go-Strips targets was detected against any of the tested near-neighbor respiratory pathogens. Cohen's kappa statistics ranged from 0.68 to 0.92 between results from the Biomeme SARS-CoV-2 Go-Strips and the BioFire® Respiratory Panel 2.1 in all the different sample types. Compared to the BioFire® Respiratory Panel 2.1, the Biomeme SARS-CoV-2 Go-Strips demonstrated statistically significantly lower sensitivity in 3 out of 5 sample types. Overall, our study demonstrates the Biomeme Franklin™ three9 used with the SARS-CoV-2 Go-Strips is an effective system for the detection of SARS-CoV-2 that could potentially be used in a remote or austere environment.

Association of Leu72Met Polymorphism of GHRL Gene and Gln223Arg of LEPR Gene With Hunger, Satiety, Biochemical, and Anthropometric Variables in Adults From Western Mexico

ObjectivesTo determine the association of Leu72Met single nucleotide polymorphism (SNP) of GHRL gene and Gln223Arg SNP of LEPR gene with hunger, satiety, biochemical, and anthropometric variables in adults from Western Mexico. MethodsQuasi-experimental study design with 132 participants of which 109 were women. Inclusion criteria were age 18–25 years old, BMI 18.5–24.9 kg/m2, 10 hours of fasting, and have the habit of eating breakfast. Exclusion criteria were subjects with a diagnosed disease, vegetarians or vegans, use of drugs that alter appetite or for weight loss, food allergies, elite athletes. Subjects were summoned twice. In the first one, medical history and anthropometric measurements were realized. A week later vital signs were taken, and blood sampling was obtained in fasting and at 120′ post breakfast. Anthropometrics and biochemical measurements were done with the InBody 370 and Vitros 350 analyzer, respectively. SNP’s were analyzed using the TaqMan® allelic discrimination assay in a real-time PCR thermocycler. Five visual analog scales to assess hunger, fullness, satiety, desire to eat, and prospective consumption were applied in fasting, after breakfast, and at 30′, 60′, 90′, and 120′ post-ingestion. Breakfast had an energy content of 526.5 kcal (36% lipids, 43% carbohydrates, and 21% protein). All variables were analyzed among genotypes considering the dominant model. Data were analyzed in SPSS version 20.0. Normality distribution was assessed with the Shapiro-Wilk test. Student T-test was applied for related (intra) or independent (inter) groups, respectively. ResultsBMI of participants was 22.0 ± 2.0 kg/m2 with a mean age of 20.6 ± 2.0 years. At 60′ post-ingestion hunger was lower and at 120′ glucose levels were lower in Leu72Leu carriers than in Leu72Met/Met72Met carriers. In fasting, total cholesterol, LDL-c, triglycerides, and desire to eat were higher in subjects with Gln223Gln genotype than in Gln223Arg/Arg223Arg genotype carriers. ConclusionsThe GHRL SNP was associated with higher hunger and glucose in the postprandial state; contrary, the LEPR SNP was associated with lower lipids levels and less desire to eat in fasting. Genetic variants could be involved with hunger, satiety, and metabolism biomarkers. Funding Sources“PROINPEP” and “PRO-SNI” from University of Guadalajara.

SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12.

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

Foot-and-mouth disease virus detection on a handheld real-time polymerase chain reaction platform.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that requires rapid control. Early detection is critical but transportation of samples to laboratory delays testing. Sensitive and specific field-deployable assays are therefore desirable. Real-time reverse transcription polymerase chain reaction (RRT-PCR) and RRT-loop-mediated isothermal amplification assays for FMDV on portable platforms have been described but none of these are handheld. In this report, we have evaluated a handheld Biomeme two3™ Real-Time PCR Thermocycler (two3) as a field-deployable platform for FMDV RRT-PCR targeting the 3D gene segment. Two3's performance was compared with the laboratory-based reference assay on the ABI7500 platform. RNA extraction using a rapid Biomeme proprietary sample prep technology (M1) was compared with MagMax RNA extraction. Two3 successfully detected FMDV isolates for six serotypes (O, A, Asia 1, SAT 1, 2 and 3). Serotype C was excluded since it has not been detected in the field since 2004. The limits of detection for serial 10-fold dilutions of cell culture isolates were equal or one log different between two3 and ABI7500. Furthermore, two3 detected FMDV RNA in multiple sample types including serum, vesicular fluid, tissue suspensions, oral fluid, oral and nasal swabs. Two3 also detected FMDV RNA directly in vesicular fluid and other samples without prior RNA extraction. Comparison of the time to first detection of a positive result in serial samples in MagMax RNA extraction/ABI7500 (MgMx/ABI) system vs. M1 RNA extraction/Two3 system revealed similar or slightly better analytical sensitivity for the MgMx/ABI system. Overall, RNA extraction by M1 yielded good results and FMDV RNA detection on two3 was not significantly different from the ABI7500. Therefore, two3 could potentially enable sensitive penside detection of FMDV within an hour using M1-extracted RNA or direct testing of vesicular fluid and swabs without RNA extraction thereby ensuring prompt implementation of appropriate control measures.

Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia.

To develop T m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5'-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52×10-5 and 4.88×10-5ng/μL samples of assemblage A and F standard plasmids. The T m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.

Type 2 diabetes associated variants of KCNQ1 strongly confer the risk of cardiovascular disease among the Saudi Arabian population.

Genome-wide association studies have identified several loci associated with an increased risk for cardiovascular disease (CVD) and type 2 diabetes (T2D). Polymorphisms within the KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1) gene are consistently associated with T2D in a number of populations. The current study was undertaken to evaluate the association of 3 polymorphisms of KCNQ1 (rs2237892, rs151290 and rs2237895) with T2D and/or CVD. Patients diagnosed with either T2D (320 patients), CVD (250 patients) or both (60 patients) and 516 healthy controls were genotyped by TaqMan assay run on a real time PCR thermocycler. A statistically significant association was found for SNPs rs151290 (OR = 1.76; 95%CI = 1.02-3.05; p = 0.0435) and rs2237895 (OR = 2.49; 95%CI = 1.72-3.61; p < 0.0001) with CVD. SNP rs151290 (OR = 7.43; 95%CI = 1.00-55.22; p = 0.0499) showed a strong association in patients with both T2D and CVD. None of the SNPs showed any significant association with T2D. Haploview analysis showed that the ACC (rs151290, rs2237892 and rs2237895) haplotype is the most significant risk allele combination for CVD, while CCA is the most significant risk haplotype for co-morbidity with T2D. KCNQ1 polymorphism at SNPs rs151290 and rs2237895 is strongly associated with CVD in this population, but presented no association with T2D.

Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment.

Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument

In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95min, as the pre-PCR preparation, including DNA extraction, required an additional 45min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods.

Stability of p53 homologs.

Most proteins have not evolved for maximal thermal stability. Some are only marginally stable, as for example, the DNA-binding domains of p53 and its homologs, whose kinetic and thermodynamic stabilities are strongly correlated. Here, we applied high-throughput methods using a real-time PCR thermocycler to study the stability of several full-length orthologs and paralogs of the p53 family of transcription factors, which have diverse functions, ranging from tumour suppression to control of developmental processes. From isothermal denaturation fluorimetry and differential scanning fluorimetry, we found that full-length proteins showed the same correlation between kinetic and thermodynamic stability as their isolated DNA-binding domains. The stabilities of the full-length p53 orthologs were marginal and correlated with the temperature of their organism, paralleling the stability of the isolated DNA-binding domains. Additionally, the paralogs p63 and p73 were significantly more stable and long-lived than p53. The short half-life of p53 orthologs and the greater persistence of the paralogs may be biologically relevant.

Lactobacillus rhamnosus GG Treatment Potentiates Intestinal Hypoxia-Inducible Factor, Promotes Intestinal Integrity and Ameliorates Alcohol-Induced Liver Injury

Gut-derived endotoxin is a critical factor in the development and progression of alcoholic liver disease (ALD). Probiotics can treat alcohol-induced liver injury associated with gut leakiness and endotoxemia in animal models, as well as in human ALD; however, the mechanism or mechanisms of their beneficial action are not well defined. We hypothesized that alcohol impairs the adaptive response-induced hypoxia-inducible factor (HIF) and that probiotic supplementation could attenuate this impairment, restoring barrier function in a mouse model of ALD by increasing HIF-responsive proteins (eg, intestinal trefoil factor) and reversing established ALD. C57BJ/6N mice were fed the Lieber DeCarli diet containing 5% alcohol for 8 weeks. Animals received Lactobacillus rhamnosus GG (LGG) supplementation in the last 2 weeks. LGG supplementation significantly reduced alcohol-induced endotoxemia and hepatic steatosis and improved liver function. LGG restored alcohol-induced reduction of HIF-2α and intestinal trefoil factor levels. In vitro studies using the Caco-2 cell culture model showed that the addition of LGG supernatant prevented alcohol-induced epithelial monolayer barrier dysfunction. Furthermore, gene silencing of HIF-1α/2α abolished the LGG effects, indicating that the protective effect of LGG is HIF-dependent. The present study provides a mechanistic insight for utilization of probiotics for the treatment of ALD, and suggests a critical role for intestinal hypoxia and decreased trefoil factor in the development of ALD.

Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

We propose a screening method for compounds affecting growth and germination in Candida albicans using a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using P(ACT1)-GFP and P(HWP1)-GFP reporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R2 > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.

Single nucleotide polymorphism detection at the Hypothenemus hampeiRdl gene by allele-specific PCR amplification with Tm-shift primers

The resistant Rdl allele for dieldrin insecticide was detected on the Hypothenemus hampei populations from Colombia using conventional PCR methods. Based on this sequence, a melting temperature ( T m) shift genotyping method that relies on allele-specific PCR is described for insecticide resistance-associated single nucleotide polymorphism (SNP) at the H. hampei Rdl gene. The method reported here uses GC-rich tails of unequal length attached to allele-specific primers containing 3′ terminal bases that correspond to SNP allelic variants. Specific PCR products are identified by inspection of a melting curve on a real-time PCR thermocycler using SYBR Green DNA binding dye. Resistant and susceptible alleles resulted in specific PCR products with T m of 83.3 ± 0.1 °C and 86.0 ± 0.2 °C, respectively. The Rdl T m-shift genotyping method is a new method to identify the Rdl gene in the coffee berry borer H. hampei, the principal pest of coffee that in general show low genetic diversity and very few genetic strategies for control of this pest have been developed. The method supplies a high-throughput tool for dieldrin resistance-associated SNP diagnostic in the coffee berry borer which will be useful for resistance-management strategies and as genetic marker in the colombian insect populations for genetics research.

Real‐time fluorescence‐based detection of furanocoumarin photoadducts of DNA

Real-time fluorescence detection systems were adapted to identify DNA adducts formed by photogenotoxic phytochemicals. Two assays were developed: the first was based on quantitative polymerase chain reaction (qPCR) while the second used thermal denaturation and renaturation (D-R). Both assays employed yeast DNA, the fluorescent dye SYBR Green and a real-time PCR thermocycler. The furanocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), psoralen, angelicin and imperatorin, and the furanochrome khellin, were tested for adduct forming ability with up to 2 h of UVA light exposure (lambda = 320-400 nm). The known bifunctional compounds, 8-MOP, 5-MOP and psoralen, were inferred to form biadducts here based on both D-R and qPCR assays, as expected from previous research. The known monofunctional compound angelicin was used as a negative control and did not form biadducts based on either assay. Two compounds of unknown functional specificity, imperatorin and khellin, were determined to be positive and negative for biadduct activity, respectively. Detection of biadducts with 8-MOP, 5-MOP, psoralen and imperatorin, but not angelicin or khellin, was further verified by temperature gradient gel electrophoresis. The fluorescence methods improve and expand upon existing assays to monitor DNA adducts.

Competitive PCR–ELISA protocols for the quantitative and the standardized detection of viral genomes

Competitive PCR-ELISA combines competitive PCR with an ELISA to allow quantitative detection of PCR products. It is based on the inclusion of an internal standard competitor molecule that is designed to differ from the target by a short sequence of nucleotides. Once such a competitor molecule has been designed and constructed, target and competitor sequences are concurrently PCR-amplified, before hybridization to two different specific probes and determination of their respective OD values by ELISA. The target can be quantified in relation to a titration curve of different dilutions of the competitor. The competitor can alternatively be used at a unique optimal concentration to allow for standardized detection of the target sequence. PCR-ELISA can be performed in 1 d in laboratories without access to a real-time PCR thermocycler. This technique is applied in diagnostics to monitor the course of infections and drug efficacy. Competitive PCR-ELISA protocols for the quantitative and for the standardized detection of parvovirus B19 are detailed here as an example of the technique.

Qualitative PCR–ELISA protocol for the detection and typing of viral genomes

PCR is an established technique providing rapid and highly productive amplification of specific DNA sequences. The demand for equally rapid, sensitive and objective methods to achieve detection of PCR products has led to the coupling of PCR with ELISA. PCR-ELISA involves direct incorporation of labeled nucleotides in amplicons during PCR-amplification, their hybridization to specific probes and hybrid capture-immunoassay in microtiter wells. PCR-ELISA is performed in 1 d and is very flexible, with the ability to process simultaneously up to 96 or 384 samples. This technique is potentially automatable and does not require expensive equipment, and thus can be fundamental in laboratories without access to a real-time PCR thermocycler. PCR-ELISA has mainly been used to detect infectious agents, including viruses, bacteria, protozoa and fungi. A PCR-ELISA protocol for the qualitative detection of papillomavirus genomes and simultaneous typing of different genotypes are detailed here as an example of the technique.

In Cultured Astrocytes, p53 and MDM2 Do Not Alter Hypoxia-inducible Factor-1α Function Regardless of the Presence of DNA Damage

A principal molecular mechanism by which cells respond to hypoxia is by activation of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Several studies describe a binding of p53 to HIF-1alpha in a protein complex, leading to attenuated function, half-life, and abundance of HIF-1alpha. However, these reports almost exclusively utilized transformed cell lines, and many employed transfection of p53 or HIF-1alpha plasmid constructs and/or p53 and HIF-1alpha reporter constructs as surrogates for endogenous protein activity and target expression, respectively. Thus, it remains an open and important question as to whether p53 inhibits HIF-1alpha-mediated transactivation of endogenous HIF-1alpha targets in nontransformed cells. After determining in primary astrocyte cultures the HIF-1alpha targets that were most dependent on HIF-1alpha function, we examined the effect of the loss of p53 function either alone or in combination with MDM2 on expression of these targets. Although p53 null astrocyte cultures resulted in markedly increased HIF-1alpha-dependent target expression compared with controls, this altered expression was determined to be the result of increased cell density of p53 null cultures and the accompanying acidosis, not loss of p53 protein. Although activation of p53 by DNA damage induced p53 target expression in astrocytes, it did not alter hypoxia-induced HIF-1alpha target expression. Finally, a combined loss of MDM2 and p53 did not alter HIF-1alpha target expression compared with loss of p53 alone. These data strongly suggest that p53 and MDM2 do not influence the hypoxia-induced transactivation of HIF-1alpha targets, regardless of p53 activation, in primary astrocytes.

A simple fluorimetric method for the estimation of DNA?DNA relatedness between closely related microorganisms by thermal denaturation temperatures

Determination of whole-genome DNA-DNA similarity is today a standard technique for species delineation in microbial taxonomy. However, these studies demand hard-to-perform and time-consuming experiments. Herein, we present an easy and rapid fluorimetric method to estimate DNA-DNA relatedness between microbial strains from differences of the thermal denaturation temperatures of hybrid and homologous genomic DNA. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence. A quantitative, real-time PCR thermocycler was used to perform the experiment and obtain fluorescence determinations at increasing temperatures. The proposed method was validated by comparing species of the hyperthermophilic genera Pyrococcus and Thermococcus. The method proves to be an easy, rapid, and inexpensive alternative to estimate DNA-DNA relatedness between closely related species.

A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature.

G+C mol% content in microorganisms is one of the recommended characteristics for the standard description of bacterial species. In this study we present a novel fluorimetric method to estimate the G+C mol% content in microorganisms. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence using a real-time PCR thermocycler. Unlike most previous determinations of G+C mol%, in this study only DNA from microorganisms with an available completely sequenced genome were used to prepare the calibration curves. Calibration curves showed a linear relationship between G+C mol% content and melting temperature and they were performed both in the absence and presence of 30% formamide. This protocol proves to be a rapid and inexpensive method to estimate DNA base ratios of novel microorganisms.