Meridian is a primary manufacturer of specialized high-quality molecular reagents and offers solutions to a wide range of industries to diagnose and treat diseases, discover new therapeutics or develop tests for environmental, food and cosmetic safety.
Enhancing Isothermal Amplification with Cutting-Edge Raw Materials Enzymes, Master Mixes and Supporting Reagents for LAMP & NASBA Assays
www.MeridianLifeScience.com
Isothermal amplification has revolutionized the point-of-care diagnostic industry, paving the way for quicker and more accessible diagnoses.
At Meridian Bioscience, we are dedicated to the development of highly sensitive, specific, and fast enzymes and master mixes to support the next generation of sensitive and affordable POC and high throughput MDx tests.
Isothermal amplification (including HAD, MCA, NASBA, and LAMP) is a DNA amplification technique that has played an increasingly important role in molecular diagnostics. It offers some advantages over qPCR in terms of sample-to-answer time and compatibility with low energy consuming devices, making it ideal for field-deployable diagnostics. Isothermal amplification assays have been used for numerous applications including the detection of pathogens such as salmonella and malaria, GM crop contamination, and forensics to detect human DNA. Isothermal Amplification Top 4 critical features in isothermal amplification that impact assay performance
• SPEED: Reduces time-to-results (TTR) and increases assay throughput • SENSITIVITY: Enables amplification of low copy-DNA or RNA templates
Loop-mediated isothermal amplification (LAMP) is one of the most common isothermal amplification methods used. However, alternative techniques such as nucleic acid sequence-based amplification (NASBA) is gaining popularity as it has the advantage of directly amplifying RNA at a lower temperature (typically around 41°C) without the need for a thermocycler or a separate reverse transcription step. Unlike LAMP which requires 4 to 6 primers, NASBA uses only two primers, which makes its primer design less complex and easier to optimize. Meridian’s LAMP and NASBA enzyme and master mixes provide state-of-the-art solutions for isothermal amplification, advancing molecular assay development. • TOLERANCE TO INHIBITORS: Enzymes with high tolerance to PCR inhibitors, improving assay robustness and reliability . • COMPATIBILITY: Assay efficiency and sensitivity is maximized when assay components such as buffer systems are optimized. For compatibility with lyophilization, the chemistry needs to be carefully optimized under glycerol-free conditions, taking into account excipients and stabilizers while ensuring no loss of performance.
LAMP
A powerful method for rapidly amplifying DNA with proven results
Loop mediated isothermal amplification (LAMP), uses four to six primers that recognize several specific regions in the target DNA. The reaction is initiated by a strand-displacement polymerase, such as Bst DNA polymerase and two specially designed primers, which form loop structures to facilitate subsequent rounds of amplification, producing high levels of DNA. LAMP reactions are very robust and inhibitor-tolerant, producing extremely large amounts of DNA that can be visualized by the eye (e.g., as a precipitate or color change). Specifically, 1 to 10 copies of DNA can be amplified to 10 9 to 10 10 copies under one hour, producing assays with excellent sensitivity and specificity.
Forward internal primer (FIP)
LAMP Reaction Workflow
*RT-LAMP requires an inital reverse transciption step to generate DNA from teh RNA template
“
Backward internal primer (BIP)
LAMP is ideal for detection of pathogens (bacteria, viruses & parasites) and genetic mutations; RT-LAMP is also used for viral RNA detection. ”
Meridian’s Bst Enzyme & LAMP Master Mix Solutions
Meridian’s Bst polymerases are designed for isothermal amplification assays and are supplied as stand-alone enzymes or pre-optimized in master mixes, for the ultimate flexibility in assay design. Our glycerol-free formulation, combined with our inhibitor tolerant buffer, is specifically suited for increasing assay robustness or establishing a direct detection workflow, avoiding the need for nucleic acid extraction. For even greater tolerance and ease of assay development, Meridian developed Specimen-specific TM inhibitor tolerant LAMP master mixes, designed to enable direct amplification from crude lysates or inhibitor-rich samples. In addition, these mixes can be used as wet mixes or dried by lyophilization or air-drying to create room-temperature stable assays. Utilizing a dried assay format not only suits POC applications but also maximizes the volume of sample to be analyzed, increasing the LOD of the test. Meridian’s innovative and ready-to-use LAMP master mixes facilitate assay chemistry optimization and overall accelerate assay development. They are ideal for developing ultra-sensitive, rapid, and simple assays, perfect for increasing a test’s competitive edge and capturing value faster in the rapidly growing POC market.
www.meridianbioscience.com/lifescience
LAMP
SPEED: Superior specificity and quicker time-to- results (TTR) detection for viral and bacterial targets
SENSITIVITY: Superior assay sensitivity and reproducibility with lower TTR and delayed NTC
60
60
50
50
40
40
30
30
20
20
10
10
0
0
Sample
NTC
Sample
NTC
Sample
NTC
Sample
NTC
Sample
NTC
Sample
NTC
Sample
NTC
Sample
NTC
Sample
NTC
Sample
NTC
HBV
HSV2
EBV
T. vaginalis
M. pneumoniae
HCV
HIV-1
DENGUE 1
WNV
Zika
Meridian Lyo-Ready Direct DNA LAMP Blood | NEB WarmStart ® | Thermo SuperScript ™
INHIBITOR TOLERANCE: High inhibitor tolerance and fast amplification to deliver quicker time-to- results (TTR) in inhibitor-rich samples. SuperScript ™ IV RT-LAMP Master Mix ( yellow ) using the same primers and DNA/RNA for five different targets as shown in the graph. Error bars represent the standard deviation across three technical replicates. For more detailed information, refer to the product flyer. The performance of Lyo-Ready Direct LAMP Blood Mix (MDX124, blue ) were compared against NEB WarmStart ™ LAMP kit ( grey ) and Thermo
The performance of Lyo-Ready Direct LAMP Urine Mix (MDX154, Blue ) were compared against NEB WarmStart ™ LAMP kit ( grey ) and Thermo SuperScript ™ IV RT-LAMP Master Mix ( yellow ) using the same primers and DNA/RNA for five different targets as shown in the graph. Error bars represent the standard deviation across three technical replicates. For more detailed information, refer to the product flyer.
COMPATIBILTIY: Compatible with lyophilization without any loss in sensitivity and speed
ADV
HSV2
50
70 60 50 40 30 20 10 0
70 60 50 40 30 20 10 0
40
30
20
10
0
1000 copies/rxn
500 copies/rxn
100 copies/rxn 10 copies/rxn
0% inhibitor
30% saliva
30% UTM+ Sputum
0% inhibitor
30% saliva
30% UTM+ Sputum
30% VTM
NTC
30% VTM
NTC
Meridian Lyophilized | NEB WarmStart®
Meridian Lyophilized | Meridian Air-dried | NEB WarmStart®
Lyophilized Lyo-Ready Direct DNA LAMP Saliva (MDX134, blue ) was compared to liquid NEB WarmStart ™ ( orange ) for their inhibitor tolerance to a range of inhibitors in amplification reactions detecting inactivated Adenovirus (ADV) or Herpes simplex virus 2 (HSV-2). Error bars represent the standard deviation across four technical replicates. For more detailed information, refer to the product information sheet.
1,000, 500, 100 and 10 copies of Emesvirus (MS2) were amplified using Air- Dryable ™ RNA/DNA LAMP (MDX118) in an air-dried ( red ) format, liquid ( blue ) format, or using NEB WarmStart ™ ( orange ) using the same primers and probes. Error bars represent standard deviation over 4 technical replicates. Fore more detailed information, refer to the product flyer.
NASBA
A preferred choice for easy isothermal assay development, lower temperature cycling, and detection of low concentrations of RNA
NASBA is an isothermal amplification method that is able to amplify and detect RNA. This technique is particularly useful in clinical virology due to its rapid amplification, high sensitivity, and high specificity, but also in POC applications where portability is critical . NASBA utilizes two primers, known as the “sense” and “antisense” primers that are designed to target a region of interest. One of these primers includes the promoter sequence for T7 RNA polymerase. The RNA is reverse transcribed to cDNA. The RNA in the cDNA-RNA hybrid is destroyed by RNase H activity and dsDNA is produced by the reverse transcriptase. This template then gets transcribed to RNA by T7 RNA polymerase and exponential amplification results.
NASBA Reaction Workflow
“
NASBA is great for highly specific amplification direcly from RNA targets at the constant and lower temprature, ideal for detecting very low concentrations of RNA is crucial, such as in viral load quantification or early detection of RNA viruses ”
www.meridianbioscience.com/lifescience
NASBA
Meridian’s NASBA Enzyme Solutions Meridian’s NASBA enzymes are designed for isothermal amplification assays and are supplied at high concentrations for flexibility in assay design. Our glycerol-free formulations offer superior performance and provide the added capability of lyophilization, granting users the flexibility to store reaction mixtures at room temperature or produce diagnostic assays with miniaturized reaction components, making them specifically suited to microfluidic or cartridge based assays. NASBA reactions consist of a reverse transcriptase, T7 RNA polymerase, and RNase H with two oligonucleotide primers. Molecular beacon probes can be used for detection as they are highly specific to their target RNA and form a stable hybrid. Although NASBA can only detect one target at a time, the technique is extremely sensitive, and ideal for clinical diagnostics, particularly for infectious diseases.
SPEED: High specificity of detection with Meridian’s NASBA enzymes under 1 hour with no detectable NTCs
HIV-1 RNA
Primers and a molecular beacon probe were designed for HIV-1 RNA and used in a nucleic acid sequence-based amplification (NASBA) reaction using Glycerol-Free T7 RNA Polymerase (HC) (MDX201), Glycerol-Free RNase H (HC) (MDX202) and AMV reverse transcriptase, using 1,000 copies of an HIV-1 RNA target.
HIV-1 RNA | NTC
SENSITIVITY: Sensitive detection with Meridian’s NASBA enzymes down to 10 copies of RNA under 1 hour
SARS-CoV 2 RNA
HIV-1 RNA
0 10 20 30 40 50
0 10 20 30 40
1000 copies 100 copies 1000 copies 100 copies
10 copies 10 copies
1000 copies 100 copies 1000 copies 100 copies
10 copies 10 copies
Primers and a molecular beacon probe were designed for HIV-1 RNA and SARS COV-2 RNA and used in NASBA reactions, with a 1,000 copy, 100 copy and 10 copy serial dilution of using of the RNA targets. The results demonstrate the high sensitivity of NASBA, being able to detect down to 10 copies per reaction in under 1 hour. This detection is highly specific sensitive and consistent, making NASBA ideal technology for the next generation of molecular point of care testing assay design.
Ordering Information LAMP Master Mixes & Enzymes
NASBA Master Mixes & Enzymes
Ready-to-Use Master Mixes
Ready-to-Use Master Mixes
Air-Dryable ™ LAMP and RT-LAMP, 4X
Lyo-Ready Direct RNA NASBA Blood
MDX119 & MDX118
MDX220
NEW
Lyo-Ready Direct RNA NASBA Saliva
MDX097 & MDX108
MDX221
Lyo-Ready LAMP and RT-LAMP, 4X
NEW
Lyo-Ready Direct RNA NASBA Stool
MDX124 & MDX125
MDX222
Lyo-Ready Direct DNA and RNA/DNA LAMP Blood, 4x
COMING SOON
Lyo-Ready Direct RNA NASBA Urine
MDX134 & MDX135
MDX223
Lyo-Ready Direct DNA and RNA/DNA LAMP Saliva, 4x
COMING SOON
Enzymes
MDX144 & MDX145
Lyo-Ready Direct DNA and RNA/DNA LAMP Stool, 4x
Glycerol-Free T7 RNA Polymerase (HC, 1,000 U/uL)
MDX201
MDX154 & MDX155
Lyo-Ready Direct DNA and RNA/DNA LAMP Urine, 4x
Glycerol-Free RNase H (HC, 50 U/uL)
Enzymes
MDX202
Glycerol-Free AMV Reverse Transcriptase (HC)
Bst DNA Polymerase (8U/ μ L)
MDX012
MDX209
NEW
Buffers
High Conc. Glycerol-Free Bst (100U/ μ L)
MDX018
Lyo-Ready NASBA Buffer
MDX210
Buffers
NEW
Enzyme Dilution Buffer
MDX078
Bst Reaction Buffer
MDX076
Inhibitor-Tolerant Bst Buffer
MDX019
Supporting Reagents
MDX083
dUTP, 100mM (Sodium)
MDX216
Glycerol-Free UDGase
dNTP Mix, 40mM (Sodium)
MDX096
NEW
MDX084
VLP-RNA Extraction Control Red
MDX217
Glycerol-Free Thermolabile UDGase
dNTP Mix, 100mM (Sodium)
MDX068
NEW
MDX063
VLP-RNA Extraction Control Orange
MDX044
MMLV-RT
dATP, 100mM (Sodium)
MDX069
MDX066
qPCR Extraction Control Red
MDX117
55C MMLV-RT
dTTP, 100mM (Sodium)
MDX026
MDX064
qPCR Extraction Control Orange
MDX056
RNase Inhibitor
dCTP, 100mM (Sodium)
MDX027
MDX065
MDX067
dNTP Mix, 10mM (Sodium)
dGTP, 100mM (Sodium)
www.meridianbioscience.com/lifescience
About Meridian
Meridian is a fully integrated life science company that develops, manufactures, markets, and distributes a broad range of innovative diagnostic products and critical raw materials. We are dedicated to developing and delivering better solutions that give answers with speed, accuracy, and simplicity that redefine the possibilities of life from discovery to diagnosis. As the Life Science division of Meridian, our focus is on developping long standing relationship with supporting immunological and molecular test manufacturers through our original raw materials for human, animal, plant, and environmental applications. The large portfolio of antigens, antibodies, blockers, molecular enzymes, nucleotides, and optimized mixes for qPCR and isothermal amplification applications are designed to simplify assay design and optimization and enable better testing overall. We strive to provide our customers with solutions they need when they need them – from novel antigens and antibodies to major disease outbreaks such as Zika and SARS-CoV-2 to pioneering the market with our innovative air-dried qPCR/RT-qPCR mixes. We take pride in providing our customers with unparalleled support, customer service, and quality.
Ordering information:
Meridian Life Science, Inc. 5171 Wilfong Road Memphis, TN 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223 Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.meridianbioscience.com/lifescience
Scan QR code to download a digital copy
Connect with us:
ISO 13485 Certified
07/24
Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8Powered by FlippingBook