Simultaneous Detection of Metalloprotease Activities in Complex Biological Samples Using the PrAMA (Proteolytic Activity Matrix Assay) Method

Abstract We combined the amplification refractory mutation system (ARMS™) and fluorescence polarization (FP) to give a homogeneous genomic DNA genotype analysis method. Oligonucleotide probes labeled with the fluorescein dyes fluorescein isothiocyanate and 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein and the rhodamine dye 6-carboxyrhodamine were included in amplification mixes and were annealed to PCR products after amplification. Hybridization was accompanied by an increase in the FP of the probe. We demonstrated homogeneous genotyping by analyzing human DNA samples for ΔF508 mutation status of the cystic fibrosis transmembrane conductance regulator gene. The genotypes determined with the method described herein were in full agreement with those obtained by the conventional application of ARMS. We also demonstrated the simultaneous detection of two PCR products in a single reaction. The assay method described is homogeneous and so obviates the necessity to open reaction vessels after amplification. This therefore eliminates PCR carryover contamination.

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Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities

Integrative Biology ◽

10.1039/c0ib00083c ◽

2011 ◽

Vol 3 (4) ◽

pp. 422-438 ◽

Cited By ~ 55

Author(s):

Miles A. Miller ◽

Layla Barkal ◽

Karen Jeng ◽

Andreas Herrlich ◽

Marcia Moss ◽

...

Keyword(s):

Simultaneous Determination ◽

Proteolytic Activity ◽

Matrix Analysis ◽

Activity Matrix

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[27] Simultaneous detection of tocopherols and tocotrienols in biological samples using HPLC-coulometric electrode array

Methods in Enzymology - Redox Cell Biology and Genetics Part A ◽

10.1016/s0076-6879(02)52029-2 ◽

2002 ◽

pp. 326-332 ◽

Cited By ~ 23

Author(s):

Sashwati Roy ◽

Mika Venojarvi ◽

Savita Khanna ◽

Chandan K. Sen

Keyword(s):

Biological Samples ◽

Simultaneous Detection ◽

Electrode Array

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A smartphone microscope method for simultaneous detection of (oo)cyst of Cryptosporodium and Giardia

10.1101/2020.04.09.035147 ◽

2020 ◽

Author(s):

Retina Shrestha ◽

Rojina Duwal ◽

Sajeev Wagle ◽

Samiksha Pokhrel ◽

Basant Giri ◽

...

Keyword(s):

Water Samples ◽

Simultaneous Detection ◽

Field Of View ◽

Assay Method ◽

Gastrointestinal Disorders ◽

Giardia Cyst ◽

White Led ◽

Major Health ◽

Microscope Imaging ◽

Illumination Source

AbstractGastrointestinal disorders caused by ingestion of (oo)cysts of Cryptosporodium and Giardia is one of the major health problems in developing countries. We developed a smartphone based microscopic assay method to screen (oo)cysts of Cryptosporodium and Giardia contamination in vegetable and water samples. We used sapphire ball lens as the major imaging element to modify a smartphone as a microscope. Imaging parameters such as field of view and magnification, and image contrast under different staining and illumination conditions were measured. The smartphone microscope method consisting of ball lens of 1 mm diameter, white LED as illumination source and Lugols’s iodine staining provided magnification and contrast capable of distinguishing (oo)cysts of Crypstopsporodium and Giardia in the same sample. The analytical performance of the method was tested by spike recovery experiments. The spiking recovery experiments performed on cabbage, carrot, cucumber, radish, tomatoes, and water resulted 26.8±10.3, 40.1±8.5, 44.4±7.3, 47.6±11.3, 49.2 ±10.9, and 30.2±7.9% recovery for Cryptosporodium, respectively and 10.2±4.0, 14.1±7.3, 24.2±12.1, 23.2±13.7, 17.1±13.9, and 37.6±2.4 % recovery for Giardia, respectively. These recovery results were found to be similar when compared with the commercial brightfield and fluorescence microscopes. We tested the smartphone microscope system for detecting (oo)cysts on 7 types of vegetable (n=196) and river water (n=18) samples. Forty two percent vegetable and thirty-nine percent water samples were found to be contaminated with Cryptosporodium oocyst. Similarly, thirty one percent vegetable and thirty three percent water samples were contaminated with Giardia cyst. This study showed that the developed method can be a cheaper alternative for simultaneous detection of (oo)cysts in vegetable and water samples.

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RT-RC-PCR: a novel and highly scalable next-generation sequencing method for simultaneous detection of SARS-COV-2 and typing variants of concern

10.1101/2021.03.02.21252704 ◽

2021 ◽

Author(s):

Christopher J Mattocks ◽

Daniel Ward ◽

Deborah JG Mackay

Keyword(s):

Reverse Transcription ◽

Limit Of Detection ◽

Simultaneous Detection ◽

Assay Method ◽

Nasopharyngeal Swab ◽

Qualitative Detection ◽

Reverse Complement ◽

Sequencing Method ◽

Polymerase Chain ◽

The Uk

We describe a novel assay method: reverse-transcription reverse-complement polymerase chain reaction (RT-RC-PCR), which rationalises reverse transcription and NGS library preparation into a single closed tube reaction. By simplifying the analytical process and cross-contamination risks, RT-RC-PCR presents disruptive scalability and economy while using NGS and LIMS infrastructure widely available across health service, institutional and commercial laboratories. We present a validation of RT-RC-PCR for the qualitative detection of SARS-CoV-2 RNA by NGS. The limit of detection is comparable to real-time RT-PCR, and no obvious difference in sensitivity was detected between extracted nasopharyngeal swab (NPS) RNA and native saliva samples. The end point measurement of RT-RC-PCR is NGS of amplified sequences within the SARS-CoV-2 genome; we demonstrated its capacity to detect different variants using amplicons containing delH69-V70 and N501Y, both of which emerged in the UK Variant of Concern B.1.1.7 in 2020. In summary, RT-RC-PCR has potential to facilitate accurate mass testing at disruptive scale and cost, with concurrent detection of variants of concern.

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Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds

Access Microbiology ◽

10.1099/acmi.0.000211 ◽

2021 ◽

Vol 3 (3) ◽

Author(s):

Aya Ahmad Elnegery ◽

Wafaa Kamel Mowafy ◽

Tarek Ahmed Zahra ◽

Noha Tharwat Abou El-Khier

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Proteolytic Activity ◽

Virulence Factors ◽

Biofilm Formation ◽

Type Species ◽

Assay Method ◽

Content Type ◽

Burn Wounds ◽

Link Type

Background. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection. Purpose. To estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes. Methods. A cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR. Results. In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain. Conclusions. QS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

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Identification and Distribution of Sterols, Bile Acids, and Acylcarnitines by LC–MS/MS in Humans, Mice, and Pigs—A Qualitative Analysis

Metabolites ◽

10.3390/metabo12010049 ◽

2022 ◽

Vol 12 (1) ◽

pp. 49

Author(s):

Ambrin Farizah Babu ◽

Ville Mikael Koistinen ◽

Soile Turunen ◽

Gloria Solano-Aguilar ◽

Joseph F. Urban ◽

...

Keyword(s):

Mass Spectrometry ◽

Bile Acid ◽

Bile Acids ◽

Biological Samples ◽

Simultaneous Detection ◽

Reversed Phase ◽

Proximal Colon ◽

Low Concentrations ◽

Human Stool ◽

Mouse Ileum

Sterols, bile acids, and acylcarnitines are key players in human metabolism. Precise annotations of these metabolites with mass spectrometry analytics are challenging because of the presence of several isomers and stereoisomers, variability in ionization, and their relatively low concentrations in biological samples. Herein, we present a sensitive and simple qualitative LC–MS/MS (liquid chromatography with tandem mass spectrometry) method by utilizing a set of pure chemical standards to facilitate the identification and distribution of sterols, bile acids, and acylcarnitines in biological samples including human stool and plasma; mouse ileum, cecum, jejunum content, duodenum content, and liver; and pig bile, proximal colon, cecum, heart, stool, and liver. With this method, we detected 24 sterol, 32 bile acid, and 27 acylcarnitine standards in one analysis that were separated within 13 min by reversed-phase chromatography. Further, we observed different sterol, bile acid, and acylcarnitine profiles for the different biological samples across the different species. The simultaneous detection and annotation of sterols, bile acids, and acylcarnitines from reference standards and biological samples with high precision represents a valuable tool for screening these metabolites in routine scientific research.

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