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Molecular and Cellular Probes

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https://www.readbyqxmd.com/read/29224776/development-of-a-sybr-green-i-real-time-pcr-for-detection-and-quantitation-of-orthopoxvirus-by-using-ectromelia-virus
#1
Wenyu Cheng, Xiaobing He, Huaijie Jia, Guohua Chen, Cong Wang, Jun Zhang, Zhizhong Jing
Ectromelia virus (ECTV) is the causative agent of mousepox, which has devastating effects in laboratory-mouse colonies and causes economic loss in biomedical research. More importantly, ECTV has been extensively used as an excellent model for studies of the pathogenesis and immunobiology of human smallpox. A rapid and sensitive SYBR Green I-based real-time PCR assay was developed and used for the detection and quantitation of orthopoxvirus by using ECTV in this study. Primers targeted to the highly conserved region of major core protein P4b gene of orthopoxvirus were designed and the standard plasmid was constructed...
December 7, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29179987/identification-of-genes-involved-in-the-four-stages-of-colorectal-cancer-gene-expression-profiling
#2
Guiling Shi, Yijia Wang, Chunze Zhang, Zhenying Zhao, Xiuying Sun, Shiwu Zhang, Jinling Fan, Cunxia Zhou, Jihong Zhang, Huijuan Zhang, Jun Liu
BACKGROUND: Colorectal cancer (CRC) is a common cancer with high morbidity and mortality. However, its molecular mechanism is not clear, nor the genes related to CRC stages. METHODS: Gene expression data in CRC and healthy colorectal tissues were obtained from gene expression omnibus. Limma package was used to identify the differentially expressed genes (DEGs) between control and CRC (stage I, II, III, and IV), obtaining 4 DEG sets. VennPlex was utilized to find all DEGs and intersection DEGs...
November 24, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29175285/development-of-hybeacon%C3%A2-probes-for-specific-mrna-detection-using-body-fluids-as-a-model-system
#3
Beccy Stafford-Allen, Nick Dawnay, Erin K Hanson, Glyn Ball, Ambika Gupta, Stephen Blackman, David J French, Nicola Duxbury, Jack Ballantyne, Simon Wells
HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA profiling. This work explores their use for the identification of expressed gene sequences through mRNA profiling. The use of mRNA is becoming increasingly common in forensic casework to identify body fluids on evidence items, as it offers higher specificity and fewer false positives than current chemical presumptive testing methods...
November 22, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29170100/genetic-differentiation-and-phylogeny-of-27-sheep-populations-based-on-structural-gene-loci
#4
Mingxun Li, Hailei Xia, Dan Chen, Dejun Ji, Tsunoda Kenji, Rui Li, Xiangxiang Liao, Yongjiang Mao, Wei Sun, Rongqing Geng, Zhangping Yang
To explore the genetic divergence and phylogeny of Chinese indigenous sheep breeds, in the current study, we analyzed the polymorphisms of 5 structural loci in ten sheep populations, including Sishui Fur, Sunite, Wurank, Bayinbuluke, Altay, Small-Tailed Han, Wadi, Tan, Tong and Hu sheep. The data were then compared with those from an additional 13 Asian and 4 European sheep populations acquired by the same experimental method. Based on the genetic distance and the results of a cluster analysis, we constructed the phylogenetic relationship of 27 populations...
November 21, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29158139/expression-and-evaluation-of-recombinant-p32-protein-based-elisa-for-sero-diagnostic-potential-of-capripox-in-sheep-and-goats
#5
Gnanavel Venkatesan, Mahesh Kumar Teli, Muthu Sankar, Amit Kumar, M Dashprakash, Sargam Arya, Aparna Madhavan, Muthannan Andavar Ramakrisnan, Awadh Bihari Pandey
The study is aimed to develop and evaluate a recombinant P32 protein based ELISA for sero-monitoring and sero-surveillance using known and suspected serum samples for capripox from sheep and goats. Truncated P32 gene of goatpox virus (with an ORF of 750 bp) was expressed in E. coli BL-21 CodonPlus (DE3) cells using pET32a vector and characterized by SDS-PAGE analysis and confirmed by western blotting as 48 KDa Poly-His tagged fusion protein. The protein was purified under denaturing conditions using 8M urea and characterized by SDS-PAGE and immunoblotting...
November 17, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29129660/microduplication-of-10q26-3-in-a-chinese-hypertriglyceridemia-patient
#6
Jing-Jing Li, Ya-Qin Chen, Liang-Liang Fan, Jie-Yuan Jin, Shuai Guo, Rong Xiang
Hypertriglyceridemia (HTG) plays an important role in the development and progression of atherosclerosis. It is inherited in an autosomal dominant pattern with a frequency of approximately 1:1,000,000 worldwide. Previous study has demonstrated that more than six genes underlie this disorder. In addition, copy number variants (CNVs) including disease-causing genes also play a crucial role in it. In this study, we have employed SNP-ARRAY chip technology to detect the pathogenic CNVs in a HTG patient who carried no meaningful mutations in HTG candidate genes...
November 9, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29129659/generation-of-conditional-acvrl1-knockout-mice-by-crispr-cas9-mediated-gene-targeting
#7
Ming Xu, Hongzhi Xu, Jian Chen, Chunjui Chen, Feng Xu, Zhiyong Qin
OBJECTIVES: This study aimed to generate mutant mice containing the Acvrl1 gene flanked with LoxP sequences to allow conditional deletion ofAcvrl1 by the LoxP/Cre system. Such mice may facilitate the development of brain arteriovenous malformation (BAVM) models. METHODS: The CRISPR/Cas9 technique was used to edit Acvrl1. Two single guide RNAs (sgRNAs) with recognition sites on intron 3 and 8 and a donor vector that was homologous with the targeted gene and contained two LoxP sequences were designed and constructed...
November 9, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29104088/a-novel-duplex-taqman-probe-based-real-time-rt-qpcr-for-detecting-and-differentiating-classical-and-variant-porcine-epidemic-diarrhea-viruses
#8
Yunfang Su, Yunchao Liu, Yumei Chen, Guangxu Xing, Huifang Hao, Qiang Wei, Yue Liang, Weitao Xie, Dongliang Li, Huimin Huang, Ruiguang Deng, Gaiping Zhang
Two different genotypes of porcine epidemic diarrhea virus (PEDV), the classical and variant strains, are classified by multiple insertions and deletions in their S genes. It is critical to detect and differentiate two genotypes in the pork industry to prevent PEDV outbreaks. In the present study, a novel duplex TaqMan RT-PCR was developed for detecting and differentiating PEDV strains in China. There was no cross-amplification between the two probes when using standard recombinant plasmids, and the specificity was further confirmed by using other seven non-PEDV swine pathogens...
November 7, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29113932/rapid-identification-of-bacillus-anthracis-by-real-time-pcr-with-dual-hybridization-probes-in-environmental-swabs
#9
Olga Bassy, Óscar Jiménez-Mateo, M Victoria Ortega, Carmen Granja, Juan Carlos Cabria
In the present study, we report the development of a real-time PCR assay for the identification of Bacillus anthracis, based on the amplification of a unique chromosomal marker, the E4 sequence, with dual hybridization probes. The assay was evaluated using a panel of ten B. anthracis strains, two B. anthracis isolates from human clinical samples, 12 B. anthracis environmental swabs and 40 non- B. anthracis strains. All 12 B. anthracis strains and clinical isolates were correctly detected, and the method did not show cross-reactions with other micro-organisms...
November 4, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29108931/binding-of-human-plasminogen-by-the-lipoprotein-lipl46-of-leptospira-interrogans
#10
Jadson V Santos, Priscila R M Pereira, Luis G V Fernandes, Gabriela Hase Siqueira, Gisele O de Souza, Antônio Souza Filho, Silvio A Vasconcellos, Marcos B Heinemann, Erica G B Chapola, Ana L T O Nascimento
Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira. Bacteria disseminate via the bloodstream and colonize the renal tubules of reservoir hosts. Leptospiral surface-exposed proteins are important targets, because due to their location they can elicit immune response and mediate adhesion and invasion processes. LipL46 has been previously reported to be located at the leptospiral outer membrane and recognized by antibodies present in serum of infected hamsters. In this study, we have confirmed the cellular location of this protein by immunofluorescence and FACS...
November 4, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29054443/development-of-an-xtag-multiplex-pcr-array-for-the-detection-of-four-avian-respiratory-viruses
#11
Feng Cong, Yujun Zhu, Xiangnan Liu, Xiuzhen Li, Meili Chen, Ren Huang, Pengju Guo
Acute respiratory tract infections are of paramount importance in the poultry industry. We developed an xTAG bead assay for the simultaneous detection and discrimination of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). The assay lacked nonspecific reactions with other common avian viruses and the limit of detection was 6.75 × 10(2)- 3.52 × 10(3)copies/μL. We examined 60 clinical specimens and found 18 positive for respiratory viruses...
October 17, 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/29050990/application-of-nucleic-acid-aptamers-for-detection-of-apple-stem-pitting-virus-isolates
#12
Beata Komorowska, Beata Hasiów-Jaroszewska, Julia Minicka
DNA aptamers (PSA-H and MT32) were applied for the detection of Apple stem pitting virus (ASPV) isolates using an Enzyme-Linked Oligonucleotide Assay (ELONA) and Western blot analysis. The specificity and effectiveness of aptamers were verified in comparison to a conventional Enzyme Linked Immunosorbent Assay (ELISA). A genetically diverse group of ASPV isolates was tested. The results showed that aptamer MT32 detected a wider range of ASPV isolates than an aptamer PSA-H and proved to be superior to commercially available monoclonal antibodies...
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28958719/recombinase-polymerase-amplification-assay-for-rapid-detection-of-porcine-circovirus-3
#13
Jianchang Wang, Yongning Zhang, Ruoxi Zhang, Qingan Han, Jinfeng Wang, Libing Liu, Ruiwen Li, Wanzhe Yuan
The objective of this study was to develop a real-time recombinase polymerase amplification (rt-RPA) assay for the rapid detection of porcine circovirus 3 (PCV3). Specific RPA primers and exo probes were designed for the cap gene of PCV3 within the conserved region of viral genome. The amplification was performed at 38 °C for 20 min. The rt-RPA was specific for PCV3, as there was no cross-reaction with other pathogens tested. Using the recombinant plasmid pUC57-PCV3 as template, the analytical sensitivity was 23 copies...
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28863892/simultaneous-detection-and-differentiation-of-human-parvovirus-b19-and-human-parvovirus-4-by-an-internally-controlled-multiplex-quantitative-real-time-pcr
#14
Junting Jia, Yadi Zhong, Yi Guo, Chaoji Huangfu, Xiong Zhao, Chi Fang, Rui Fan, Yuyuan Ma, Jingang Zhang
Human parvovirus B19 (B19V) and human parvovirus 4 (PARV4) are two parvoviruses known to infect humans and transmit through blood and plasma derived medicinal products (PDMPs). Inactivation of the two parvoviruses has proven to be difficult and nucleic acid testing (NAT) would be an efficient means to exclude viruses. In this study, an internally controlled multiplex quantitative real-time PCR (qPCR) assay for B19V and PARV4 simultaneous detection and quantification was established and evaluated. The optimized multiplex qPCR assay allowed for simultaneous detection of all of the genotypes (1-3) of B19V and PARV4, with equal limit of quantification (LOQ) of 5 copies/μL, rather than other blood-borne viruses...
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28842221/rapid-and-visual-detection-of-mycobacterium-tuberculosis-complex-using-recombinase-polymerase-amplification-combined-with-lateral-flow-strips
#15
Qinglin Ma, Houming Liu, Feidi Ye, Guangxin Xiang, Wanshui Shan, Wanli Xing
To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA...
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28826997/detection-of-skeletonema-costatum-based-on-loop-mediated-isothermal-amplification-combined-with-lateral-flow-dipstick
#16
Hai-Long Huang, Peng Zhu, Cheng-Xu Zhou, Xiao-Jun Yan, Yi-Xin Zou, Pei-Wen Lv
We developed a new assay method, which combines loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) for the rapid and special detection of the diatom Skeletonema costatum. Four groups of LAMP primers were derived from a conserved DNA sequence unique to S. costatum. The amplifications were carried out at 61, 63, and 65 °C for 60 min in various combinations by the quantitative PCR thermal cycler to confirm optimal primers and reaction temperature. The LAMP-LFD detection limit was 0...
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28803792/rapid-and-visual-detection-of-leptospira-in-urine-by-ligb-lamp-assay-with-pre-addition-of-dye
#17
Syed Atif Ali, Gurpreet Kaur, Nongthombam Boby, T Sabarinath, Khushal Solanki, Dheeraj Pal, Pallab Chaudhuri
Leptospirosis is considered to be the most widespread zoonotic disease caused by pathogenic species of Leptospira. The present study reports a novel set of primers targeting LigB gene for visual detection of pathogenic Leptospira in urine samples through Loop-mediated isothermal amplification (LAMP). The results were recorded by using Hydroxyl napthol blue (HNB), SYBR GREEN I and calcein. Analytical sensitivity of LAMP was as few as 10 leptospiral organisms in spiked urine samples from cattle and dog. LigB gene based LAMP, termed as LigB-LAMP, was found 10 times more sensitive than conventional PCR...
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28668278/synthetic-scale-up-of-a-novel-fluorescent-probe-and-its-biological-evaluation-for-surface-detection-of-staphylococcus-aureus
#18
Luke Bywaters, Lauren Mulcahy-Ryan, Mark Fielder, Alex Sinclair, Adam Le Gresley
This paper reports on the LGX fluorometric test for enzymatic MRSA/MSSA detection. It highlights the reasons rhodamines have been overlooked and also strategies to improve the synthesis of rhodamine-peptide conjugates. Evaluation of the LGX test for detection of MRSA/MSSA on surfaces is undertaken in the presence of potentially confounding E. coli and S. epidermidis for the first time.
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28666619/development-of-a-multiplex-pcr-for-identification-of-%C3%AE-hemolytic-streptococci-relevant-to-human-infections-and-serotype-distribution-of-invasive-streptococcus-agalactiae-in-thailand
#19
Anusak Kerdsin, Rujirat Hatrongjit, Shigeyuki Hamada, Yukihiro Akeda, Marcelo Gottschalk
A multiplex polymerase chain reaction (mPCR) was developed for simultaneous detection (single reaction) of genes specific to five frequent clinically relevant β-hemolytic streptococcal species: Streptococcus pyogenes (Spy1258), Streptococcus agalactiae (cfb and cpn60), Streptococcus dysgalactiae subsp. equisimilis (16S-23S intergenic spacer) , S. equi subsp. zooepidemicus (esaA and sorD), and Streptococcus anginosus group (moaC). No cross-reaction was observed with other bacterial species. This test was validated and successfully used with 725 clinical isolates involved in pathological conditions in Thailand and collected between March 2014 and December 2015...
December 2017: Molecular and Cellular Probes
https://www.readbyqxmd.com/read/28652020/development-of-a-realtime-rt-pcr-assay-for-the-rapid-detection-of-influenza-a-h2-viruses
#20
Andrey Komissarov, Artem Fadeev, Anna Kosheleva, Kseniya Sintsova, Mikhail Grudinin
Influenza and other acute respiratory infections are of great concern for public health, causing excessive morbidity and mortality throughout the world. Influenza virus A(H2N2), which caused a pandemic of so called "Asian flu" in 1957 was expelled from the human population by the new pandemic virus subtype H3N2 in 1968, however, influenza A(H2) viruses continue to circulate in wild birds and poultry. The lack of immunity in human population and the continued circulation of influenza A(H2) among animals makes emergence of a new pandemic virus possible...
October 2017: Molecular and Cellular Probes
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