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2022 ◽  
pp. 230-243
Author(s):  
Antonia Mourtzikou ◽  
Marilena Stamouli ◽  
Georgia Kalliora ◽  
Panagiotis Koumpouros ◽  
Ioanna Petraki ◽  
...  

Clinical laboratories produce test results that support the diagnosis, prognosis, and patient treatment. Test results must be relevant, accurate, and reliable for patient care. International bibliographic data estimate that approximately 62.0% of the errors made in clinical laboratories are due to errors during the pre-analytical stage. This chapter presents a failure modes and effects analysis (FMEA) to analyze potential failure risks within the pre-analytical phase and classify them according to severity and likelihood. FMEA allows molecular laboratories to lower costs and drive better outcomes through high-quality nucleic acid extraction, sensitive detection, and accurate quantification. RT-PCR technology continues to be the gold standard for the clinical detection of SARS-CoV-2 RNA in individuals suspected of COVID-19. It is essential to use highly sensitive assays to detect active infections and reduce the likelihood of false-negative results.


2021 ◽  
Vol 12 ◽  
Author(s):  
Hanen Chelbi ◽  
Refka Jelassi ◽  
Sarra Belfkih ◽  
Amor Ben Amor ◽  
Nasreddine Saidi ◽  
...  

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia. Materials and methods: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects. Results: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls (p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects (p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer (p = 0.788, OR = 1.265, 95% CI: 0.228-7.011). Conclusion: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms.


2021 ◽  
pp. 113873
Author(s):  
Jia Yao ◽  
Yuanyuan Luo ◽  
Zhiqi Zhang ◽  
Jinze Li ◽  
Chuanyu Li ◽  
...  

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Haixia Wang ◽  
Xuechun Zhao ◽  
Hui Wang

Background. Breast cancer (BC) progression is related to the disorder of circular RNAs (circRNAs). This study aims to characterize the role of circ_0075943 in BC. Methods. Real-time fluorescent quantitative PCR (real-time PCR) technology was implemented to investigate circ_0075943, AK2 mRNA, and microRNA-141-3p levels. MTT, colony formation method, Transwell, and flow cytometry technique were adopted to investigate cell function. The connection between miR-141-3p and circ_0075943 or AK2 was confirmed by the dual-luciferase reporter gene or RNA immunoprecipitation (RIP). The influence on circ_0075943 in vivo was confirmed by animal experiments. Results. circ_0075943 was augmented in BC cell lines and tumor specimens. Dwindling of circ_0075943 could dramatically suppress the phenotype of BC cells and induce apoptosis. MiR-141-3p is a target of circ_0075943, and its repression largely reverses the influence of knocking down circ_0075943 on cell behavior. Moreover, AK2, as a target of miR-141-3p, is augmented in BC cells and specimens. AK2 overexpression could restore the phenotype of BC cells blocked by miR-141-3p redevelopment. Moreover, knocking down circ_0075943 could suppress the growth of tumors in vivo. Conclusion. The abnormal regulation of circ_0075943 participates in part of the expansion of BC by dominating the miR-141-3p/AK2 regulatory network.


2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Irshad Ahmad ◽  
Youri Lee ◽  
Nighat Nawaz ◽  
Rizwan Elahi ◽  
Israr Ali Khan ◽  
...  

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3), then amplified expression of the DYC18_RS18060-His6 protein was achieved with detection by SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins. Copyright(c)  The Authors


2021 ◽  
Vol 934 (1) ◽  
pp. 012027
Author(s):  
M Mardalisa ◽  
U M Batubara

Abstract Belulang grass (Eleusine indica) is a plant in the Poaceae family that is commonly found in the coastal area of Dumai, Riau Province. Eleusine indica is characterized by narrow leaves, concave stems that can reach up to 95 cm high and strong roots. E. indica is known to be very tolerant of its environment, including the environment contaminated with heavy metals. The ability of E. indica as a phytoremediation agent in absorbing heavy metals has been widely known as the role of metallothionein (MT) protein. MT is believed to have a function in the metal metabolism and detoxification process through the metal chelating interaction between the cysteine amino acid residues. This unique function prompted the interest to isolate the MT gene from E. indica. This method involves the isolation of genomic DNA from E. indica followed by the process of amplification of the MT gene using specific primers, namely MTFS and MTRS by polymerase chain reaction (PCR) technology. The success of the MT gene isolation process from E. indica was evidenced by the presence of a single band size of around 172 bp via the visualization process on 1% agarose gel. Furthermore, the results of the PCR product are purified for the purpose of sequencing activity. The results of sequencing analysis of the 172 bp fragment showed 99.31% identical similarity with the complete metallothionein gene from E. indica (DQ082855.1) by using the BLASTN tool, NCBI website.


2021 ◽  
Vol 8 (1) ◽  
pp. 012-021
Author(s):  
Luka Yelwa Barde ◽  
Hussaini Adamu ◽  
Grace Ifemedike Uzoma ◽  
Mohammed Bello ◽  
Mohammed Abba Danjuma

Advances in biotechnology has been the subject of praise for a decade now, this is due to techniques such as gene expression which has contributed immensely in the success of genetic engineering, medical advancement, vaccine production and agriculture. Gene expression has become a very important tool for the overall improvement of quality of life. This paper tries to look into the development of gene expression in the last forty years and to highlight how technological advancements in the study of gene expression brought about improvements as a focal point. Technological advancements associated with northern blotting, western blotting, and enzyme-linked immunosobent assays, Polymerase Chain Reaction (PCR) technology with emphasis on quantitative PCR, differential protein display technique and DNA sequencing and hybridization arrays technology with emphasis on macroarrays and microarrays in facilitating gene expression will be discussed.


2021 ◽  
Vol 22 (11) ◽  
pp. 6061
Author(s):  
Owen Higgins ◽  
Terry J. Smith

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.


2021 ◽  
Author(s):  
Shamanta Grosso ◽  
Lucia Pagani ◽  
Nilla Tosoni ◽  
Massimo Crapis ◽  
Enrico Turrini ◽  
...  

The value of blood cultures for confirming the clinical diagnosis of sepsis is suboptimal. There is growing interest in the potential of real-time PCR technology by detection of minute amounts of pathogen DNA in patient blood samples with results available within 4–6 h. Adopting a two-step approach, we evaluated the compliance of two versions of the MicrobScan assay on a total of 748 patients with suspected bloodstream infections. The results obtained with a second version of the MicrobScan assay are characterized by increased specificity (from 95.1 to 98.2%) and sensitivity (from 76.7 to 85.1), increased throughput and the possibility of simultaneously testing different kinds of samples collected from the potential sites of infection and utilizing different syndromic panels.


2021 ◽  
Vol 790 (1) ◽  
pp. 012053
Author(s):  
Omran Hassan AL-Sarhan ◽  
Abdulkhalik Alwan Mohemeed ◽  
Ahmed Yas Saeed
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