Real Time Pcr
Recently Published Documents





2021 ◽  
Vol 12 ◽  
Seung-Min Yang ◽  
Eiseul Kim ◽  
Dayoung Kim ◽  
Hyeon-Be Kim ◽  
Jiwon Baek ◽  

An accurate diagnostic method for Salmonella serovars is fundamental to preventing the spread of associated diseases. A diagnostic polymerase chain reaction (PCR)-based method has proven to be an effective tool for detecting pathogenic bacteria. However, the gene markers currently used in real-time PCR to detect Salmonella serovars have low specificity and are developed for only a few serovars. Therefore, in this study, we explored the novel unique gene markers for 60 serovars that share similar antigenic formulas and show high prevalence using pangenome analysis and developed a real-time PCR to detect them. Before exploring gene markers, the 535 Salmonella genomes were evaluated, and some genomes had serovars different from the designated serovar information. Based on these analyses, serovar-specific gene markers were explored. These markers were identified as genes present in all strains of target serovar genomes but absent in strains of other serovar genomes. Serovar-specific primer pairs were designed from the gene markers, and a real-time PCR method that can distinguish between 60 of the most common Salmonella serovars in a single 96-well plate assay was developed. As a result, real-time PCR showed 100% specificity for 199 Salmonella and 29 non-Salmonella strains. Subsequently, the method developed was applied successfully to both strains with identified serovars and an unknown strain, demonstrating that real-time PCR can accurately detect serovars of strains compared with traditional serotyping methods, such as antisera agglutination. Therefore, our method enables rapid and economical Salmonella serotyping compared with the traditional serotyping method.

Krishnapriya Umashankar ◽  
J. Selvaraj ◽  
Pratibha Ramani

Background: Oral squamous cell cancer (OSCC) develops as a result of the accumulation of many genetic mutations that are influenced by genetic predisposition. Upon acquisition of genetic predisposition, the precancerous cells will transform into malignant cells culminating into carcinomas. Advances in genetic research over the past few decades have rendered early detection possible. Aim: To compare the gene expression of Pi3 Kinase, AKT and mTOR in OSCC and to correlate the expression levels of these molecules with the survival in OSCC patients. Also to understand the role of Pi3 Kinase pathway in OSCC progression thereby attempting targeted therapy in OSCC patients. Materials and Methods: 10 OSCC samples as well as normal healthy samples were collected and RNA isolation was done using RNA easy kit from Qiagen (Valencia, CA), and then subjected to cDNA synthesis using Human TGF-β1, Human GSK-3β and Human Pi3 kinase primers. Real time PCR was performed using gene specific primers at 40 cycles. The results were retrieved, tabulated and analyzed. Results: The current research results revealed that there were up regulation of mRNA expression in The PI3K/AKT/mTOR in OSCC patients than in healthy individuals. On comparison, mTOR showed highest mRNA expression levels than AKT and PI3K. Conclusion: Overexpression of Pi3 kinase, AKT, mTOR plays a crucial role in progression of oral cancer and targeting Pi3 kinase/mTOR pathways could be a novel and targeted approach for OSCC.

2021 ◽  
Abdolkhalegh Deezagi ◽  
Bahar Ghorbani

Abstract Angiogenesis is an important process in tumor growth and metastasis and vascular endothelial growth factor (VEGF) plays an important role in this process. Several VEGF inhibitors have been developed as anticancer agents including humanized monoclonal antibodies, and various small molecules. The aim of this work was to investigate the effect of combination of VEGF siRNA and Avastin on breast cancer MCF-7 cell line behavior. For this purpose, the cells were treated with different concentrations of Avastin and/or VEGF siRNA and their combination. The cell survival and cell proliferation were assayed by cell counting, trypan blue and MTT tests. The cell migration was assayed by scratching test. VEGF expression was assayed by RT and real-time PCR and ELISA methods. Results indicated the significant increase in cell death following treatment with Avastin (50% cell death at 100 µg/ml). Cell death with VEGF siRNA transfection was lower than Avastin, however, it was significant. This result in VEGF siRNA + Avastin (100 µg/ml) treatment was greater compared to treatment with each of these compounds alone (47%). Scratching results also showed the synergic effect of VEGF siRNA and Avastin (57% decrease). Real-time PCR results showed that Avastin at concentrations of ≥ 50 µg/ml led to 2.5 to 7.5-fold decrease in VEGF expression levels. Also, treatment with VEGF siRNA led to 15.5-fold decrease in VEGF expression. Finally, VEGF expression following VEGF siRNA + Avastin treatment led to a significant 47.5-fold decrease in VEGF expression. It could be concluded that combination of VEGF siRNA and Avastin have a more significant impact on the inhibition of cell growth and migration and it can probably be used as an effective therapeutic approach.

2021 ◽  
Vol 37 (7) ◽  
Ayesha Nayyar ◽  
Suhaib Ahmed

Objective: To optimize and evaluate a real time PCR of Single Nucleotide Polymorphism by SYBR Green method for detection of donor chimerism after haematopoietic stem cell transplantation. Methods: This descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Oct 2017 - Dec 2019. A total of twenty patients of post haematopoietic stem cell transplant with various haematological disorders were studied to see the status of donor chimerism by using SNP real time PCR using SYBR Green method and short tandem repeat PCR. These patients had undergone allogeneic HSCT from HLA-matched sibling donors at Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre. Results: Real time PCR using SYBR Green was able to detect significant amount of chimerism in all 20 patients having undergone HSCT. Regarding precision of the real time PCR assay the mean value of donor chimerism was 94.1% (SD 3.96) and by STR PCR it was 95.1% (SD 1.41). The assay was found to be sensitive with a detection limit of <1%. Conclusion: Our results demonstrate that SNP analysis by SYBR Green real time PCR may be used for the evaluation of chimerism status in patients having undergone HSCT with a sensitivity of <1%. Hence donor chimerism by this sensitive method can be used in monitoring of chimerism in post-transplant patients with various haematological disorders. doi: How to cite this:Nayyar A, Ahmed S. Donor Chimerism Study by Single Nucleotide Polymorphism using SYBR green based Real Time PCR. Pak J Med Sci. 2021;37(7):---------. doi: This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Biomeditsina ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. 10-16
V. N. Karkischenko ◽  
N. V. Petrova ◽  
V. V. Slobodenyuk ◽  
N. A. Laryushina

Mini-pigs are an adequate biomodel for characterizing the processes of working capacity and endurance, as well as for conducting investigations in the field of sports medicine. For this kind of research, we propose gene targets from the families of cytokine and sirtuin proteins: IL-6, HMGB1, TNF (genes responsible for the synthesis of proteins belonging to the cytokine group) and SIRT (transferase from the family of sirtuin proteins). The SIRT 1 gene presents particular interest as an activator of mitochondrial activity during exercise. Real-time PCR systems were created, allowing assessment of the effect of various drugs on laboratory mini-pigs in preclinical studies.

2021 ◽  
Vol 15 (9) ◽  
pp. e0009765
Sudha Chaturvedi ◽  
Tanya R. Victor ◽  
Anuradha Marathe ◽  
Ketevan Sidamonidze ◽  
Kelly L. Crucillo ◽  

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.

2021 ◽  
Vol 14 (2) ◽  
Trần Thanh Tú ◽  
Nguyễn Thị Vân Anh ◽  
Phùng Thị Bích Thủy ◽  
Bùi Thị Huyền ◽  
Nguyễn Thị Thanh Phúc ◽  

Đặt vấn đề/ Mục tiêu: Viêm nhiễm virus hợp bào hô hấp RSV (Respiratory Syncytial virus) là một trong những nguyên nhân phổ biến gây bệnh đường hô hấp. Nghiên cứu này được thực hiện nhằm bước đầu khảo sát tác dụng hỗ trợ điều trị của sản phẩm probiotic LiveSpo® Navax dạng nước chứa bào tử lợi khuẩn Bacillus thế hệ LS-III ở nồng độ cao trên đối tượng trẻ em bị bệnh đường hô hấp cấp do nhiễm RSV tại bệnh viện Nhi Trung ương. Phương pháp: Bước đầu đánh giá trên 30 bệnh nhân được chẩn đoán bị bệnh viêm tiểu phế quản do nhiễm RSV tham gia vào nghiên cứu thử nghiệm lâm sàng ngẫu nhiên có đối chứng mù. Bệnh nhân được chia ngẫu nhiên vào 2 nhóm (n =15/nhóm): nhóm sử dụng LiveSpo® Navax (nhóm Navax) và nhóm sử dụng nước muối sinh lý NaCl 0,9% (nhóm Chứng), được hướng dẫn xịt mũi với tần suất 3 lần/ngày trong 6 ngày liên tục, kết hợp với sử dụng thuốc điều trị thường quy tại bệnh viện. Bệnh nhân được tiến hành theo dõi các chỉ số lâm sàng (khò khè, khó thở, độ bão hòa oxy,...) trong suốt thời gian điều trị và được thực hiện các xét nghiệm như: (i) đo tải lượng RSV, nồng độ của B. subtilis và B. clausii ở ngày 0 và ngày 3 trong dịch tỵ hầu bằng phương pháp Real-time PCR. Kết quả: Nhóm Navax có thời gian khỏi các triệu chứng xuất tiết mũi, khó thở, ran rít, ran ẩm, rút lõm lồng ngực sớm hơn nhóm đối chứng khoảng 1 ngày. Sau 3 ngày điều trị, tải lượng RSV ở nhóm Navax ở dịch tỵ hầu của bệnh nhân giảm khoảng 300 lần, trong khi nhóm đối chứng chỉ giảm 15 lần, có liên quan tới sự có mặt của bào tử vi khuẩn B. subtilis và B. clausii ở dịch mũi bệnh nhân nhóm Navax mà vắng mặt ở nhóm đối chứng. 100% bệnh nhân sử dụng LiveSpo® Navax không có bất cứ dấu hiệu bất thường nào về rối loạn nhịp thở, mạch, kích ứng niêm mạc mũi, hay tiêu hóa. Kết luận: Đây là nghiên cứu thử nghiệm lâm sàng ở trẻ em đầu tiên trên thế giới về an toàn và tác dụng của probiotic bào tử lợi khuẩn Bacillus ở dạng xịt mũi. LiveSpo®Navax có tác dụng rút ngắn khoảng 1 ngày thời gian điều trị các triệu chứng điển hình của bệnh đường hô hấp do nhiễm RSV gây ra và làm giảm nồng độ virus hợp bào hô hấp RSV trong mũi của trẻ em hiệu quả hơn gấp 20 lần so với nước muối sinh lý. 

2021 ◽  
Vol 1 (1) ◽  
Letian Zhang ◽  
Meng Lu ◽  
Jiaxuan Lu ◽  
Ningning Wang ◽  
Zhongzhou Pan ◽  

AbstractInfluenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide. Genome structure of influenza D virus (IDV) is identical to that of influenza C virus (ICV), and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology. To date, the prevalence of ICV and IDV in China is unclear, but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential. To efficiently monitor ICV and IDV, it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research. A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed. TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed. This method exhibited good specificity and sensitivity, and the detection limit reached 1 × 101 copies/μL of plasmid standards of each pathogen. Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method. One positive sample of IDV was detected, and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing. The duplex real-time PCR detection method represents a sensitive and specific tool to detect ICV and IDV. It provides technical support for virus research and clinical diagnosis of ICV and IDV. This information will benefit animal and human health.

2021 ◽  
Jingzhu Zhou ◽  
Xiuhai Ji ◽  
yan fen ◽  
Hui ding

Abstract Background: IPF is a progressive lung disease, characterized by excessive deposition of ECM. C/EBPβ is involved in the development of pulmonary fibrosis. However, the regulation of C/EBPβ in the context of pulmonary fibrosis is not clear. The study is to identify the C/EBPβ acetylation in IPF.Methods: Lung from six IPF and six control samples were selected in this study. We investigated the expression of C/EBPβ in lungs with Immunochemistry. Moreover, the expression of C/EBPβ mRNA via Real Time-PCR and its protein expression via Western Blot were performed. Meanwhile, the levels of collagen-I and α-SMA as markers of pulmonary fibrosis were also determined by Western Blot. Furthermore, we confirmed the relationship between α-SMA and acetylated C/EBPβ by Co-Immunoprecipitation. Results: We found the elevated C/EBPβ mostly locating in fibroblast foci in lungs of IPF. And the expression of C/EBPβ RNA and protein were obviously increased in IPF (P <0.05), in which the proteins of α-SMA and collagen-I were enhanced (P <0.05). Furthermore, the stronger acetylation of C/EBPβ binging to the α-SMA gene was shown in lung fibrosis (P <0.05). Conclusions: The increased expression of C/EBPβ acetylation associated with α-SMA expression is involved in the development of pulmonary fibrosis.

2021 ◽  
Vol 15 (9) ◽  
pp. e0009782
Jason L. Cantera ◽  
Heather N. White ◽  
Mattahew S. Forrest ◽  
Oliver W. Stringer ◽  
Vicente Y. Belizario ◽  

Background Soil-transmitted helminths (STHs) are parasitic nematodes that inhabit the human intestine. They affect more than 1.5 billion people worldwide, causing physical and cognitive impairment in children. The global strategy to control STH infection includes periodic mass drug administration (MDA) based on the results of diagnostic testing among populations at risk, but the current microscopy method for detecting infection has diminished sensitivity as the intensity of infection decreases. Thus, improved diagnostic tools are needed to support decision-making for STH control programs. Methodology We developed a nucleic acid amplification test based on recombinase polymerase amplification (RPA) technology to detect STH in stool. We designed primers and probes for each of the four STH species, optimized the assay, and then verified its performance using clinical stool samples. Principal findings Each RPA assay was as sensitive as a real-time polymerase chain reaction (PCR) assay in detecting copies of cloned target DNA sequences. The RPA assay amplified the target in DNA extracted from human stool samples that were positive for STH based on the Kato-Katz method, with no cross-reactivity of the non-target genomic DNA. When tested with clinical stool samples from patients with infections of light, moderate, and heavy intensity, the RPA assays demonstrated performance comparable to that of real-time PCR, with better results than Kato-Katz. This new rapid, sensitive and field-deployable method for detecting STH infections can help STH control programs achieve their goals. Conclusions Semi-quantitation of target by RPA assay is possible and is comparable to real-time PCR. With proper instrumentation, RPA assays can provide robust, semi-quantification of STH DNA targets as an alternative field-deployable indicator to counts of helminth eggs for assessing infection intensity.

Sign in / Sign up

Export Citation Format

Share Document