scholarly journals Species Identification and Quantification of Bovine Bone Marrow from Non-Bovine Products using DNA and Protein Fingerprints

2021 ◽  
Author(s):  
Yize Guan ◽  
Nan Li ◽  
Tancheng Li ◽  
Liyuan Sun ◽  
Li Ming Cheng
Keyword(s):  
Bone Marrow ◽  
Multiplex Pcr ◽  
High Sensitivity ◽  
Bovine Bone ◽  
Assay Sensitivity ◽  
Conserved Sequence ◽  
Specific Pcr ◽  
Modified Method ◽  
Sds Page ◽  
Mitochondrial Cytochrome B

Abstract Bovine bone marrow is traditionally regarded as a highly nutritious food that has been widely used as a medicinal and healthy food for several decades in China. A large number of adulterated and counterfeit bone marrows from pigs and donkeys have been used in place of bovine bone marrow in commercial products, which are almost identical morphologically to species. Therefore, we explored a feasibility of fingerprinting of multiplex PCR gels combined with imaged SDS-PAGE gels of proteins. Three pairs of specific primers for bovine, pig and donkey were designed according to the conserved sequence in mitochondrial cytochrome b. The optimal reaction conditions for triple PCR were optimized. A modified method was used to extract total proteins and SDS-PAGE gels of proteins were set up. A three-fold PCR detection assay was successfully established to identify three species of bovine, pig and donkey. Three primers have good specificity and high sensitivity. Additionally, the assay sensitivity test confirmed that the extracted DNA concentration was the lowest of bovine bone marrow at 0.1 pg. The assay also showed 100% specificity. The electrophoretogram of SDS-PAGE showed distinct electrophoresis profiles among three species. The established fingerprint of multiplex PCR DNA gels combined with imaged SDS-PAGE gels of proteins has strong specificity and can be quickly and accurately used for the identification of bovine bone marrow. Rapid authentication of bovine bone marrow and differentiation from non-bovine products can be achieved using an improved SDS alkali denaturation method, species-specific PCR assay combined with imaged SDS-PAGE gels of proteins.

scholarly journals Specific PCR Detection of Tiger, Leopard, and Lion Ingredients from Test Samples

2011 ◽  
Vol 94 (4) ◽  
pp. 1200-1205
Author(s):  
Jijuan Cao ◽  
Junyi Xu ◽  
Ran Liu ◽  
Ke Yu ◽  
Changwen Wang
Keyword(s):  
Food Processing ◽  
Processing Condition ◽  
High Sensitivity ◽  
Animal Protection ◽  
Specific Pcr ◽  
Mitochondrial Cytochrome B ◽  
Inspection And Quarantine ◽  
Powder Samples ◽  
Effect Of Food ◽  
Pcr Method

Abstract A PCR method was developed for specifc detection of tiger, leopard, and lion DNA from test specimens for inspection and quarantine or for law-enforced animal protection. Three pairs of specifc primers were designed based on the mitochondrial cytochrome b gene of tiger, leopard, and lion and used in the PCR testing. To mimic the effect of food processing on the sensitivity of the test, the tiger muscle and bovine bonemeal powder samples were treated at 133°C for 30 min. At this processing condition, the method was sensitive enough to detect as low as 0.05% of tiger-derived ingredients from the mixed bonemeal powders. The data demonstrate that our PCR method is convenient and economic, with high sensitivity and repeatability, and can be used to detect and identify tiger, leopard, and lion ingredients from various test samples.

scholarly journals Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245175
Author(s):  
Sujintana Janesomboon ◽  
Veerachat Muangsombut ◽  
Varintip Srinon ◽  
Chatruthai Meethai ◽  
Chayada S. Tharinjaroen ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Capsular Polysaccharide ◽  
High Sensitivity ◽  
Pcr Primers ◽  
Soil Samples ◽  
Pcr Assay ◽  
Pathogenic Potential ◽  
Specific Pcr ◽  
Phylogenetic Cluster ◽  
Multiplex Pcr Assay

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil.

scholarly journals AB0050 A NOVEL METHOD FOR ISOLATION OF EXOSOMES FROM SYNOVIAL FLUID

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1057.2-1057
Author(s):  
Y. Liu ◽  
Y. Huang ◽  
Q. Huang ◽  
S. Sun ◽  
Z. Ji ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Western Blot ◽  
Biological Fluid ◽  
High Sensitivity ◽  
Protein Distribution ◽  
Sds Page ◽  
Close Relationship ◽  
Novel Method ◽  
Low Levels

Background:Exosomes in synovial fluid (SF) has a close relationship with the pathogenesis of rheumatiod arthritis. As a complex biological fluid, SF presents challenges for exosomes isolation using standard methods, such as ExoquickTM kit and ultracentrifugation.Objectives:The study aims to compared the quality of exosomes separated by ExoquickTM kit (TM), ExoquickTM kit+ExoquickTC kit (TM-TC), ultracentrifugation (UC) and TM-TC+UC(TM-TC-UC) from SF.Methods:Exosomes was separated by TM, TM-TC, UC and TM-TC-UC respectively. The size and concentrations of exosomes were detected by high sensitivity flow cytometry for nanoparticle analysis. Total protein and RNA were extracted from exosomes. SDS-PAGE was used to detect the protein distribution of exosomes. Western blot was used to examine the level of albumin and exosomes marker (TSG101 and CD81).Results:There was no statistic difference in the diameters of exosomes separated by the four methods. The concentrations of exosomes in TM, TM-TC, TM-TC-UC and UC were (5.65±0.93), (3.02±1.19), (1.67±0.25) and (4.61±0.73) *109Particles/mL. The protein concentrations of exosomes separated by the four methods were consistent with the concentrations of exosomes. SDS-PAGE showed that the protein distribution of exosomes separated by the four methods were different. Low levels of albumin were detected in TM-TC and TM-TC-UC, while high levels of albumin in TM and UC. Total RNA concentrations from exosomes in TM-TC was higher than other groups.Conclusion:TM-TC can be used to obtain higher quality exosomes from SF for the study of exosome-enriched components.References:[1]Helwa I, et al, A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents. PloS one, 2017. 12(1): p. e0170628-e0170628.Figure 1.A: SDS-PAGE showed the protein distribution of exosomes; B: the detection of albumin, TSG101 and CD81 by western blot.Disclosure of Interests:None declared

THE FATE OF BOVINE BONE MARROW STROMAL CELLS IN HYDROGELS

2008 ◽  
Vol 41 ◽  
pp. S130 ◽  
Author(s):  
Stephan Zeiter ◽  
Marije van der Werf ◽  
Keita Ito
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Bovine Bone

A new subset of small stem cells in bovine bone marrow stromal cell populations

2019 ◽  
Vol 120 (8) ◽  
pp. 13881-13892
Author(s):  
Shaohui Pan ◽  
Yu‐Chen Chen ◽  
Ning Zhao ◽  
Xiang Feng ◽  
Dan‐Dan Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Cell Populations ◽  
Bovine Bone ◽  
Marrow Stromal Cell

scholarly journals RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 827
Author(s):  
Paula Rodrigues de Almeida ◽  
Ana Karolina Antunes Eisen ◽  
Meriane Demoliner ◽  
Fernando Rosado Spilki
Keyword(s):  
Reverse Transcription ◽  
Tissue Concentration ◽  
Critical Role ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Assay Sensitivity ◽  
Robust Detection ◽  
Detection Techniques ◽  
Mosquito Pool

Zika virus (ZIKV) is an important arbovirus, responsible for recent outbreaks of Guillain Barré Syndrome and Congenital Zika Syndrome (CZS). After thousands of CZS cases, ZIKV is under constant surveillance in Brazil. Reliable and robust detection techniques are required to minimize the influence of host inhibitors from clinical samples and mosquito pool samples. Reverse transcription Digital Polymerase Chain Reaction (RT-dPCR) is a technique that allows the accurate quantification of DNA targets with high sensitivity, and it is usually less affected by inhibitors than RT-qPCR. This study aimed to assess the influence of mosquito tissue, RNA extraction and cDNA synthesis in ZIKV PCR detection. Samples containing 0, 3 and 10 mosquitoes were spiked with ZIKV MR766 and serially diluted prior to RNA extraction and RT-dPCR for ZIKV. Two reverse transcription protocols were tested. Assay sensitivity allowed the detection of 1.197 copies/µL. A higher correlation between dilution factor and target quantification was observed in 10 mosquito pool samples. The lower quantification in samples diluted without mosquitoes highlights the critical role of the reverse transcription step in RNA detection, since it could be attributed to reverse transcriptase variable performance in samples with low overall RNA concentration. The results in mosquito pools indicate that mosquito tissues do not inhibit ZIKV RT-dPCR, and the RT-dPCR technique has good sensitivity and robustness for ZIKV detection in mosquito pool samples regardless of mosquito tissue concentration.

scholarly journals Mixed hematopoietic chimerism after allogeneic transplantation with lymphocyte-depleted bone marrow is not associated with a higher incidence of relapse

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1367-1372 ◽  
Author(s):  
A Schattenberg ◽  
T De Witte ◽  
M Salden ◽  
J Vet ◽  
B Van Dijk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Allogeneic Bone Marrow Transplantation ◽  
High Sensitivity ◽  
Lymphocyte Culture ◽  
Mixed Chimerism ◽  
Allogeneic Bone ◽  
Red Cell ◽  
Marrow Graft ◽  
Mixed Chimeras

Abstract Using red cell phenotyping, cytogenetic analysis of blood lymphocytes, chromosome studies of bone marrow cells, and restriction fragment length polymorphism (RFLP) studies of peripheral blood cells, we demonstrated a high number of mixed chimeras after allogeneic bone marrow transplantation (BMT). Donor marrow from HLA-A, -B, and -DR identical, mixed lymphocyte culture (MLC) nonreactive siblings was depleted of 98% of lymphocytes using counterflow centrifugation. Thirty- two of 48 recipients (67%) appeared to be mixed chimeras at 6 months after transplantation. The high number of mixed chimeras is probably a result of lymphocyte depletion of the marrow graft and the high sensitivity of red cell phenotyping for the demonstration of minor cell populations (at levels as low as 0.01%). The probability of relapse- free survival from 6 months to 4 years after BMT was 85% for the mixed chimeras and 65% for the complete donor chimeras. We conclude that in this study, mixed chimerism is not associated with a higher incidence of relapse.

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