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Archives of Razi Institute Aug 2023Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the...
Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the disease and eliminating it completely from livestock production has been associated with numerous problems. This project aimed to identify the molecular cause of pebrine disease in silkworms using a sensitive, specific, and accurate method. To this purpose, a 136 bp fragment was selected based on the partial SSU rDNA sequence, and a pair of primers was designed. Afterward, using the conventional polymerase chain reaction (PCR) method, the target fragment was amplified and sequenced. After that, to determine the detection sensitivity, using the Real-Time PCR method, 5-fold serial dilutions of DNA were prepared, and the last dilution that produced a fluorescent signal was considered the minimum detection limit. All tests were performed in duplicates. Based on the results of the sensitivity test, the standard curve including Ct values and DNA concentration was used for analysis. Moreover, 80 unknown samples examined by light microscope were evaluated using conventional PCR and Real-Time PCR. Both PCR results showed no amplification for the negative control samples. The findings demonstrated that the lowest detection limit for was less than 6 pg of DNA, while, this amount was 8 ng for conventional PCR. Out of 80 samples examined, 55, 60, and 62 samples were positive for light microscope, conventional PCR, and Real-Time PCR methods, respectively. The findings suggested that the Real-Time PCR method had a higher ability to detect the causative agent of pebrine disease than the conventional PCR method, and both methods were superior to light microscopy. Therefore, due to the fewer steps and higher accuracy of Real-Time PCR, it can be introduced as a suitable method for diagnosing pebrine disease.
Topics: Animals; Bombyx; Microsporidiosis; Real-Time Polymerase Chain Reaction; DNA Primers; DNA
PubMed: 38226388
DOI: 10.32592/ARI.2023.78.4.1185 -
Journal of Insect Science (Online) Sep 2023The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from...
The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ± 3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ± 4.57 ng/µl) and a microwave-based method (119.26 ± 3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ± 0.02, 1.79 ± 0.01, and 1.87 ± 0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.
Topics: Animals; Culicidae; Culex; DNA; Polymerase Chain Reaction; DNA Primers
PubMed: 37804500
DOI: 10.1093/jisesa/iead080 -
Advanced Science (Weinheim,... Oct 2023The COVID-19 outbreak has caused public and global health crises. However, the lack of on-site fast, reliable, sensitive, and low-cost reverse transcription polymerase...
The COVID-19 outbreak has caused public and global health crises. However, the lack of on-site fast, reliable, sensitive, and low-cost reverse transcription polymerase chain reaction (RT-PCR) testing limits early detection, timely isolation, and epidemic prevention and control. Here, the authors report a rapid mobile efficient diagnostics of infectious diseases via on-chip -RT-quantitative PCR (RT-qPCR): MEDIC-PCR. First, the authors use a roll-to-roll printing process to accomplish low-cost carbon-black-based disposable PCR chips that enable rapid LED-induced photothermal PCR cycles. The MEDIC-PCR can perform RT (3 min), and PCR (9 min) steps. Further, the cohort of 89 COVID-19 and 103 non-COVID-19 patients testing is completed by the MEDIC-PCR to show excellent diagnostic accuracy of 97%, sensitivity of 94%, and specificity of 98%. This MEDIC-PCR can contribute to the preventive global health in the face of a future pandemic.
Topics: Humans; Reverse Transcriptase Polymerase Chain Reaction; COVID-19; Sensitivity and Specificity; Polymerase Chain Reaction; Communicable Diseases; COVID-19 Testing
PubMed: 37587764
DOI: 10.1002/advs.202302072 -
Scientific Reports Oct 2023Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection, and several qRT-PCR kits have been...
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection, and several qRT-PCR kits have been established targeting different genes of the virus. Due to the high mutation rate of these genes, false negative results arise thus complicating the interpretation of the diagnosis and increasing the need of alternative targets. In this study, an alternative approach for the detection of SARS-CoV-2 viral RNA targeting the membrane (M) gene of the virus using qRT-PCR was described. Performance evaluation of this newly developed in-house assay against commercial qRT-PCR kits was done using clinical oropharyngeal specimens of COVID-19 positive patients. The limit of detection was determined using successive dilutions of known copies of SARS-CoV-2 pseudovirus. The M gene based assay was able to detect a minimum of 100 copies of virus/mL indicating its capacity to detect low viral load. The assay showed comparable accuracy, sensitivity and specificity with commercially available kits while detecting all the variants efficiently. The study concluded that the in-house M gene based assay might be an effective alternative for the currently available commercial qRT-PCR kits.
Topics: Humans; SARS-CoV-2; COVID-19; Reverse Transcriptase Polymerase Chain Reaction; COVID-19 Testing; Sensitivity and Specificity; RNA; RNA, Viral; Real-Time Polymerase Chain Reaction
PubMed: 37789078
DOI: 10.1038/s41598-023-43204-9 -
Chinese Medical Journal Mar 2024Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be...
BACKGROUND
Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.
METHODS
The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed.
RESULTS
The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts.
CONCLUSIONS
This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.
Topics: Humans; HIV-1; DNA, Viral; Reproducibility of Results; Polymerase Chain Reaction; HIV Infections; Real-Time Polymerase Chain Reaction
PubMed: 38433332
DOI: 10.1097/CM9.0000000000003081 -
Yeast (Chichester, England) Nov 2023Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome...
Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.
Topics: Saccharomyces cerevisiae; Debaryomyces; Polymerase Chain Reaction; Gene Targeting; Biotechnology
PubMed: 37870109
DOI: 10.1002/yea.3902 -
Scientific Reports Dec 2023In the last decade, clinical studies have investigated the clinical relevance of circulating cell-free-DNA (ccfDNA) as a diagnostic and prognosis tool in various...
In the last decade, clinical studies have investigated the clinical relevance of circulating cell-free-DNA (ccfDNA) as a diagnostic and prognosis tool in various diseases including cancers. However, limited knowledge on ccfDNA biology restrains its full development in the clinical practice. To improve our understanding, we evaluated the impact of the circadian rhythm on ccfDNA release in healthy subjects over a 24-h period. 10 healthy female subjects underwent blood sampling at 8am and 20 healthy male subjects underwent serial blood sampling (8:00 AM, 9:00 AM, 12:00 PM, 4:00 PM, 8:00 PM, 12:00 AM, 4 AM (+ 1 Day) and 8 AM (+ 1 Day)). We performed digital droplet-based PCR (ddPCR) assays to target 2 DNA fragments (69 & 243 bp) located in the KRAS gene to determine the ccfDNA concentration and fragmentation profile. As control, half of the samples were re-analyzed by capillary miniaturized electrophoresis (BIAbooster system). Overall, we did not detect any influence of the circadian rhythm on ccfDNA release. Instead, we observed a decrease in the ccfDNA concentration after meal ingestion, suggesting either a post-prandial effect or a technical detection bias due to a higher plasma load in lipids and triglycerides. We also noticed a potential effect of gender, weight and creatinine levels on ccfDNA concentration.
Topics: Humans; Male; Female; Healthy Volunteers; Cell-Free Nucleic Acids; Prognosis; Polymerase Chain Reaction; DNA; Circadian Rhythm
PubMed: 38065990
DOI: 10.1038/s41598-023-47851-w -
Poultry Science Aug 2023Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant...
Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant injury to small poultry and tremendous economic damage to the poultry sector. This research aims to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) approach to simultaneously determine these important viral pathogens. The conserved segment of various viral genetic sequences was used to design and synthesize specific primers. Moreover, as positive controls, recombinant vectors were synthesized in this investigation. The d-optimal approach was used to improve PCR conditions in this investigation. Positive controls and clinical samples were used to assess the m-PCR assay's specificity, sensitivity, repeatability, and reproducibility. According to the sensitivity test findings, the m-PCR technique could generate the 8 target genes from viral genomes using 1 × 102. In addition, 8 viral pathogens were detected from the infected samples. The findings also suggest that live animal oral swabs were not significantly different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for analyzing both types of samples. The suggested m-PCR test may detect and evaluate viral infection in birds with excellent specificity, sensitivity, and throughput.
Topics: Animals; Poultry; Reverse Transcriptase Polymerase Chain Reaction; Reproducibility of Results; Reverse Transcription; Chickens; Sensitivity and Specificity; Bird Diseases; Virus Diseases; Respiratory Tract Infections; Polymerase Chain Reaction; Multiplex Polymerase Chain Reaction; Poultry Diseases
PubMed: 37354617
DOI: 10.1016/j.psj.2023.102852 -
Advanced Science (Weinheim,... Nov 2023DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot...
DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot be actively processed in DNA pools. An active DNA data editing process is developed using splint ligation in a droplet-controlled fluidics (DCF) system. DNA fragments of discrete sizes (100-500 bps) are synthesized for droplet assembly, and programmed sequence information exchange occurred. The encoded DNA sequences are processed in series and parallel to synthesize the determined DNA pools, enabling random access using polymerase chain reaction amplification. The sequencing results of the assembled DNA data pools can be orderly aligned for decoding and have high fidelity through address primer scanning. Furthermore, eight 90 bps DNA pools with pixel information (png: 0.27-0.28 kB), encoded by codons, are synthesized to create eight 270 bps DNA pools with an animation movie chip file (mp4: 12 kB) in the DCF system.
Topics: DNA; Polymerase Chain Reaction
PubMed: 37755129
DOI: 10.1002/advs.202303197 -
Frontiers in Cellular and Infection... 2023Candidate Phyla Radiation (CPR) and more specifically Candidatus Saccharibacteria (TM7) have now been established as ubiquitous members of the human oral microbiota....
INTRODUCTION
Candidate Phyla Radiation (CPR) and more specifically Candidatus Saccharibacteria (TM7) have now been established as ubiquitous members of the human oral microbiota. Additionally, CPR have been reported in the gastrointestinal and urogenital tracts. However, the exploration of new human niches has been limited to date.
METHODS
In this study, we performed a prospective and retrospective screening of TM7 in human samples using standard PCR, real-time PCR, scanning electron microscopy (SEM) and shotgun metagenomics.
RESULTS
Using Real-time PCR and standard PCR, oral samples presented the highest TM7 prevalence followed by fecal samples, breast milk samples, vaginal samples and urine samples. Surprisingly, TM7 were also detected in infectious samples, namely cardiac valves and blood cultures at a low prevalence (under 3%). Moreover, we observed CPR-like structures using SEM in all sample types except cardiac valves. The reconstruction of TM7 genomes in oral and fecal samples from shotgun metagenomics reads further confirmed their high prevalence in some samples.
CONCLUSION
This study confirmed, through their detection in multiple human samples, that TM7 are human commensals that can also be found in clinical settings. Their detection in clinical samples warrants further studies to explore their role in a pathological setting.
Topics: Female; Humans; Prospective Studies; Retrospective Studies; Bacteria; Microbiota; Real-Time Polymerase Chain Reaction
PubMed: 37577371
DOI: 10.3389/fcimb.2023.1195679