scholarly journals 3D-Printed Microfluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

2018 ◽  
Author(s):  
Yonghee Kim ◽  
Jinyeop Lee ◽  
Sungsu Park
Keyword(s):  
Genomic Dna ◽  
Molecular Detection ◽  
Immunomagnetic Separation ◽  
Automated System ◽  
Dna Purification ◽  
Clinical Samples ◽  
Microfluidic Platform ◽  
Silica Bead ◽  
3D Printed ◽  
Magnetic Silica

Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica bead (MSB)-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a 3D-printed microfluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two microchannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The device was able to preconcentrate very low concentrations of pathogens and extract their genomic DNA in 10 mL of 10% blood within 30 min, and thus allowed polymerase chain reaction (PCR) and quantitative PCR to detect 1 colony forming unit of Escherichia coli O157:H7 in 10% blood. The results suggest that the 3D-printed microfluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample.

scholarly journals A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

Micromachines ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 472 ◽  
Author(s):  
Yonghee Kim ◽  
Jinyeop Lee ◽  
Sungsu Park
Keyword(s):  
Genomic Dna ◽  
Molecular Detection ◽  
Three Dimensional ◽  
Immunomagnetic Separation ◽  
Automated System ◽  
Dna Purification ◽  
Clinical Samples ◽  
Silica Bead ◽  
3D Printed ◽  
Magnetic Silica

Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1–1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample.

scholarly journals A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics

2021 ◽  
Vol 7 (5) ◽  
pp. eabc7802
Author(s):  
Kai Shi ◽  
Shiyi Xie ◽  
Renyun Tian ◽  
Shuo Wang ◽  
Qin Lu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genomic Dna ◽  
Reaction Network ◽  
Clinical Samples ◽  
Great Promise ◽  
Base Specificity ◽  
Nucleotide Mutation ◽  
Positive Feedback Circuit ◽  
Stringent Target ◽  
And Function

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

scholarly journals Molecular detection of Brucella spp. from broth culture of clinical samples in Nigeria: Its role in vaccine quality control

2009 ◽  
Vol 8 (21) ◽  
pp. 5661-5665 ◽  
Author(s):  
F Antiabong J ◽  
Yakubu B ◽  
A Owolodun O ◽  
Bertu W ◽  
A Ocholi R
Keyword(s):  
Quality Control ◽  
Molecular Detection ◽  
Broth Culture ◽  
Clinical Samples

scholarly journals Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽  
2010 ◽  
Vol 21 (1) ◽  
Author(s):  
Nono Carsono ◽  
Sri Nurlianti ◽  
Inez Nur Indrayani ◽  
Ade Ismail ◽  
Tri Joko Santoso ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Oryza Sativa ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Oryza Sativa L ◽  
Dna Purification ◽  
Coding Region ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak.  Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

A Hybrid Paper and 3D Printed Microfluidic Platform for Electrochemical Detection of Ag-Labeled Magnetic Particles

2021 ◽  
Vol MA2021-02 (57) ◽  
pp. 1858-1858
Author(s):  
Charuksha Walgama ◽  
Nicole Pollok ◽  
Lisa Boatner ◽  
Richard Crooks
Keyword(s):  
Electrochemical Detection ◽  
Magnetic Particles ◽  
Microfluidic Platform ◽  
3D Printed

Automated fluorometer/photometer system for homogeneous immunoassays.

1983 ◽  
Vol 29 (9) ◽  
pp. 1628-1634 ◽  
Author(s):  
T M Li ◽  
S P Robertson ◽  
T H Crouch ◽  
E E Pahuski ◽  
G A Bush ◽  
...  
Keyword(s):  
Fixed Point ◽  
Photon Counting ◽  
Reference Method ◽  
Automated System ◽  
Clinical Samples ◽  
System A ◽  
Sample Throughput ◽  
Software Updates ◽  
Specific Proteins ◽  
Blood Analytes

Abstract A fully automated bench-top clinical analyzer (OPTIMATE TM; Ames/Gilford) performs homogeneous fluorescent immunoassays, colorimetric immunoassays, and determinations of routine blood analytes; drugs, enzymes, metabolites, specific proteins, and hormones in serum. Unique features include a combination fluorescence/absorbance aspirating thermocuvette, a photon-counting fluorometer/photometer, a multi-reagent distribution valve to dispense as many as three reagents plus buffer, and a user-replaceable programmable memory cartridge for software updates. We have evaluated the performance of OPTIMATE substrate-labeled fluorescent immunoassays for gentamicin, tobramycin, amikacin, theophylline, phenytoin, phenobarbital, primidone, carbamazepine, and quinidine with this automated system. A sample throughput of 92 samples per hour is achieved by reading fixed-point fluorescence results every 39 s after an initial 4-min reaction period. Precision studies indicate typical CVs of less than or equal to 6% for mid-range controls. Standard curves can be reused for as long as two weeks before recalibration. With clinical samples, results by the OPTIMATE procedure correlated well (r greater than or equal to 0.97) with those by a reference method.

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