Use of Direct PCR Technique Without DNA Extraction in Confirmation Test for Salmonella typhimurium Bacteria on Meatball Samples

The use of direct PCR technique without DNA extraction in the confirmation test for Salmonella typhimurium ATCC 14028 bacteria on meatball samples was carried out in the Food and Drug molecular biology testing laboratory Administration in Gorontalo. The basis of this research is to have an impact on economic value in carrying out the confirmation test for S. typhimurium ATCC 14028, where testing is carried out conventionally, namely DNA extraction, which requires a large amount of money. Hence, it is necessary to innovate to modify the testing phase so that it is more effective and efficient. The purpose of this study was to see whether the direct PCR technique without DNA extraction can be done for the confirmation test of S. typhimurium ATCC 14028 on meatball samples. This study's sample consisted of 20 types of meatball samples spiked with S. typhimurium ATCC 14028 cultures. The method used in this study was qPCR analysis using the SYBR Green method. Data analysis was carried out based on 2 main criteria: (1) Ct analysis and (2) Tm analysis. Real-time PCR analysis results obtained Ct values in the range 14.14 - 15.20 with an average of 14.82 and Tm values 85.20 - 86.30 with an average of 85.79. Based on these data, it can be concluded that using direct PCR can be used for testing confirmation of S. typhimurium ATCC 14028 on meatball samples.

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Detection of Salmonella typhimurium ATCC 14028 in Powder Prepared Traditional Medicines Using Real-Time PCR

Borneo Journal of Pharmacy ◽

10.33084/bjop.v4i3.1838 ◽

2021 ◽

Vol 4 (3) ◽

pp. 178-183

Author(s):

Alfi Sophian ◽

Ratna Purwaningsih ◽

Muindar Muindar ◽

Eka Putri Juniarti Igirisa ◽

Muhammad Luthfi Amirullah

Keyword(s):

Salmonella Typhimurium ◽

Real Time ◽

Real Time Pcr ◽

Positive Control ◽

Negative Control ◽

Pcr Analysis ◽

Direct Pcr ◽

Traditional Medicines ◽

Pcr Method ◽

Alternative Testing

The detection of Salmonella typhimurium ATCC 14028 using real-time PCR on powdered traditional medicinal products was carried out in the microbiology and molecular biology testing laboratory of the Food and Drug Administration in Gorontalo. This research aims to provide a reference for alternative testing methods in testing the products of traditional powder preparations on the market. The sample consisted of 10 traditional powder preparations spiked with positive control of S. typhimurium ATCC 14028 phase 2. The method used in the study was real-time PCR analysis using the SYBR® Green method, while DNA isolation using the direct PCR method. Data analysis was performed by analyzing the sample's melting temperature (Tm) curve and comparing it with positive control. The results showed that S. typhimurium ATCC 14028 was detected in samples at an average Tm value of 84.18°C, with ranges of 84.0-84.5°C. For positive control, the Tm value was at 85.2°C, while for the negative control, the Tm value was not detected. Based on these data, it can be concluded that S. typhimurium ATCC 14028 in traditional medicine products powder preparations can be detected using real-time PCR.

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Biological and Molecular Characteristics of a Novel Partitivirus Infecting the Edible Fungus Lentinula edodes

Plant Disease ◽

10.1094/pdis-07-16-0951-re ◽

2017 ◽

Vol 101 (5) ◽

pp. 726-733 ◽

Cited By ~ 15

Author(s):

Mengpei Guo ◽

Yinbing Bian ◽

Jinjie Wang ◽

Gangzheng Wang ◽

Xiaolong Ma ◽

...

Keyword(s):

Lentinula Edodes ◽

Control Strategies ◽

Phylogenetic Analyses ◽

Pcr Analysis ◽

Molecular Characteristics ◽

Rt Pcr ◽

Edible Fungus ◽

Qpcr Analysis ◽

Disease Diagnostics ◽

Wild Strains

A new partitivirus named Lentinula edodes partitivirus 1 (LePV1) was isolated from a diseased L. edodes strain with severe degeneration of the mycelium and imperfect browning in bag cultures. The nucleotide sequences of LePV1 dsRNA-1 and dsRNA-2 were determined; they were 2,382 bp and 2,245 bp in length, and each contained a single ORF encoding RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively. The purified virus preparation contained isometric particles 34 nm in diameter encapsidating these dsRNAs. Phylogenetic analyses showed LePV1 to be a new member of Betapartitivirus, with the RdRp sequence most closely related to Grapevine partitivirus. RT-PCR analysis showed that 27 of the 56 Chinese L. edodes core collection strains carry LePV1, with the virus being more common in wild strains than cultivated strains. In addition, qPCR analysis suggested that coinfection with L. edodes mycovirus HKB (LeV-HKB) could increase replication of the RdRp gene of LePV1. This study may be essential for the development of more accurate disease diagnostics and the formulation of control strategies for viral diseases in L. edodes.

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Direct PCR from whole blood, without DNA extraction

Nucleic Acids Research ◽

10.1093/nar/18.19.5908 ◽

1990 ◽

Vol 18 (19) ◽

pp. 5908-5909 ◽

Cited By ~ 113

Author(s):

B. Mercier ◽

C. Gaucher ◽

O. Feugeas ◽

C. Mazurier

Keyword(s):

Dna Extraction ◽

Whole Blood ◽

Direct Pcr

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Detection of Bacteria in Environmental Samples by Direct PCR Without DNA Extraction

BioTechniques ◽

10.2144/01313rr04 ◽

2001 ◽

Vol 31 (3) ◽

pp. 598-607 ◽

Cited By ~ 29

Author(s):

Kimberly A. Fode-Vaughan ◽

Charles F. Wimpee ◽

Charles C. Remsen ◽

Mary Lynne Perille Collins

Keyword(s):

Dna Extraction ◽

Environmental Samples ◽

Direct Pcr

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Key Regulatory Pathways, MicroRNAs, and Target Genes Participate in Adventitious Root Formation of Acer Rubrum L

10.21203/rs.3.rs-793059/v2 ◽

2021 ◽

Author(s):

Wenpeng Zhu ◽

Manyu Zhang ◽

Jianyi Li ◽

Hewen Zhao ◽

Kezhong Zhang ◽

...

Keyword(s):

Transcriptional Activation ◽

High Throughput Sequencing ◽

Target Genes ◽

Seedling Survival ◽

Expression Patterns ◽

Economic Value ◽

Acer Rubrum ◽

Pcr Analysis ◽

Asexual Propagation ◽

Regulatory Pathways

Abstract BackgroundAcer rubrum L. is a colorful ornamental tree with great economic value. Because this tree is difficult to root under natural conditions and the seedling survival rate is low, vegetative propagation methods are often used. Because the formation of adventitious roots (ARs) is essential for the survival of asexual propagation of A. rubrum, it is necessary to investigate the molecular regulatory mechanisms in the formation of ARs of A. ruburm. To address this knowledge gap, we sequenced the transcriptome and sRNA of the A. rubrum variety ‘Autumn Fantasy’ using high-throughput sequencing and explored changes in gene and microRNA (miRNA) expression in response to exogenous auxin treatment. ResultsWe identified 82,468 differentially expressed genes between the treated and untreated ARs, as well as 48 known and 95 novel miRNAs. We also identified 172 target genes of the known miRNAs using degradome sequencing. Two regulatory pathways (ubiquitin mediated proteolysis and plant hormone signal transduction), Ar-miR160a and the target gene ArARF10 were shown to be involved in the auxin response. We further investigated the expression patterns and regulatory roles of ArARF10 through subcellular localization, transcriptional activation, plant transformation, qRT-PCR analysis, and GUS staining. ConclusionsDifferential expression patterns indicated the Ar-miR160a-ArARF10 interaction might play a significant role in the regulation of AR formation in A. rubrum. Our study provided new insights into mechanisms underlying the regulation of AR formation in A. rubrum.

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Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in Apple

10.21203/rs.2.21019/v5 ◽

2020 ◽

Author(s):

Xiya Zuo ◽

Shixiang Wang ◽

Wen Xiang ◽

Huiru Yang ◽

Muhammad Mobeen Tahir ◽

...

Keyword(s):

Gene Family ◽

Expression Profiles ◽

Expression Patterns ◽

Economic Value ◽

Floral Transition ◽

Bimolecular Fluorescence Complementation ◽

Pcr Analysis ◽

Transcriptional Responses ◽

Genome Database ◽

Reverse Transcription Pcr

Abstract Background: Apple (Malus domestica Borkh.) is a popular cultivated fruit crop with high economic value in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple.Results: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14‑3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus.Conclusion: We identified the Md14-3-3s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3s in floral transition.

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A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta

Letters in Applied Microbiology ◽

10.1111/lam.12196 ◽

2013 ◽

Vol 58 (4) ◽

pp. 350-355 ◽

Cited By ~ 4

Author(s):

C. Biswas ◽

P. Dey ◽

S. Satpathy

Keyword(s):

Dna Extraction ◽

Rapid Detection ◽

Direct Pcr

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Determining the optimal method for DNA isolation from fruit jams

Czech Journal of Food Sciences ◽

10.17221/340/2017-cjfs ◽

2018 ◽

Vol 36 (No. 2) ◽

pp. 126-132

Author(s):

Sovová Tereza ◽

Křížová Barbora ◽

Ovesná Jaroslava

Keyword(s):

Dna Extraction ◽

Dna Isolation ◽

Extraction Methods ◽

Food Samples ◽

Pcr Analysis ◽

Uv Spectrophotometry ◽

Pcr Assay ◽

Optimal Method ◽

Ctab Method ◽

Dna Extraction Methods

DNA extraction is a crucial step in PCR analysis especially when analysing food samples that can be degraded and can potentially contain PCR-inhibiting substances. In this study, we compared the suitability of three DNA extraction methods – two kits: DNeasy® Plant Mini Kit and NucleoSpin® Food, and the CTAB method – for DNA extraction from commercial fruit jams. Fourteen jams with different contents of fruit, sugar and other additives were extracted in triplicate using the above-mentioned methods directly and after a washing step. The concentration and optical density were analysed using UV spectrophotometry and the amplifiability of the obtained DNA was evaluated using a PCR assay targeting a sequence coding for chloroplast tRNA-Leu. Samples isolated using the NucleoSpin® Food kit contained non-amplifiable DNA in eight cases, and samples isolated using the CTAB method could not be quantified. The DNeasy® Plant Mini Kit thus proved to be the most suitable method, since well-amplifiable DNA was obtained for all the analysed samples.

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