Sample-specific mixes for direct DNA or RNA isothermal amplification from saliva or sputum. Lyo-Ready Direct DNA LAMP Saliva and RNA/DNA LAMP Saliva are ready-to-use, 4x concentrated, fully optimized mixes for isothermal amplification methods such as LAMP, designed for sensitive amplification of RNA or DNA from crudely processed saliva or sputum samples.
Lyo-Ready TM Direct LAMP Saliva Mixes
Sample-specific mixes for direct DNA or RNA isothermal amplification from saliva or sputum
Lyo-Ready ™ Direct DNA LAMP Saliva and RNA/DNA LAMP Saliva are ready-to-use, 4x concentrated, fully optimized mixes for isothermal amplification methods such as LAMP, designed for sensitive amplification of RNA or DNA from crudely processed saliva or sputum samples. Saliva is ideal for use as a diagnostic specimen as it contains hormones, antibodies, growth factors, enzymes and microbes that can be used as biomarkers for disease detection. Saliva is easy-to-collect and allows non-invasive and simple sample collection/logistics, making it perfect for point of care testing. However, due to the low concentration of analytes and high concentration of inhibitors in saliva, their use in molecular diagnostics has been limited. Isothermal amplification such as LAMP is very tolerant to inhibitors and the simple workflow allows fast, sensitivity and reliability results in clinical diagnosis. Meridian’s new Lyo-Ready ™ Direct DNA LAMP and RNA/DNA LAMP Saliva mixes are the newest advancement in molecular reagent technology and combine the benefits of a highly inhibitor-tolerant mix with robust isothermal amplification to enable direct detection using crudely processed samples. The mixes are uniquely designed to overcome saliva and sputum inhibitors and they do not require a DNA or RNA purification step to concentrate and purify a patient sample. In addition, the 4x concentrated master mixes are formulated with lyo-excipients which are optimized for freeze-drying to create ambient-temperature stable assays.
By using an innovative ready-to-use isothermal master mix such as Lyo-Ready ™ Direct DNA LAMP or RNA/DNA LAMP Saliva mixes, assay development time for a new ultra-sensitive saliva isothermal assay is rapid, simple and efficient. Perfect for increasing your competitive edge and capturing value faster in the rapidly growing point-of-care market.
• Inhibitor-tolerant isothermal mix designed for direct amplification from crude saliva or sputum samples • Very fast reaction kinetics – ideal for point of care testing • Fully optimized, concentrated 4x mix– just add custom primers and probes • Suitable for detecting RNA or DNA pathogens at very low titers • Mixes can be lyophilized to create ambient- temperature stable assays Product Highlights
PRODUCT
CAT NO. VOLUME REACTIONS
5 mL 800 Rxns 50 mL 8,000 Rxns 800 Rxns 50 mL 8,000 Rxns 5 mL
Lyo-Ready ™ Direct DNA LAMP Saliva
MDX134
Lyo-Ready ™ Direct RNA/DNA LAMP Saliva
MDX135
www.MeridianLifeScience.com
High inhibitor tolerance and fast amplification to deliver quicker time-to-results (TTR) in saliva, sputum and samples containing Universal Transport Media (UTM)
Lyo-Ready ™ Direct DNA LAMP Saliva (MDX134)
CMV
ADV
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
0
0% inhibitor
30% saliva
30% UTM + Sputum
30% VTM
NTC
0% inhibitor
30% saliva
30% UTM + Sputum
30% VTM
NTC
HSV2
Primers for CMV, ADV or HSV-2 were added to Lyo-Ready ™ Direct DNA LAMP Saliva (MDX134) and lyophilized ( blue ) or added to NEB WarmStart ® ( orange ) and kept as a liquid. The mixes were amplified using inactivated Cytomegalovirus (CMV), Adenovirus (ADV) or Herpes simplex virus 2 (HSV2). Reactions were prepared in the presence of 30% human saliva, 30% Universal Transport Medium (UTM) + an artificial sputum swab and Viral Transport Medium (VTM). All reactions were incubated at 65 °C for 60 minutes, and average time to results (TTR) was measured at 1:10 of end fluorescence. Error bars represent the standard deviation across four technical replicates. Results show that MDX134 ( blue ) has a shorter TTR than NEB WarmStart ® ( orange ) amplification across all three sample types. The data suggests that MDX134 performs better with faster amplification across the range of inhibitors.
70
60
50
40
30
20
10
0
0% inhibitor
30% saliva
30% UTM + Sputum
30% VTM
NTC
Lyophilized | NEB WarmStart ®
Lyo-Ready ™ Direct RNA/DNA LAMP Saliva (MDX135)
RSV
Primers for RSV were added to Lyo-Ready ™ Direct RNA/DNA LAMP Saliva (MDX135) and lyophilized ( blue ) or added to NEB WarmStart ® ( orange ) and kept as a liquid. The mixes were amplified using inactivated respiratory syncytial virus (RSV) RNA prepared with 30% human saliva, 30% Universal Transport Medium (UTM) with/without an artificial sputum swab and Viral Transport Medium (VTM). All reactions were incubated at 65 °C for 60 minutes, and the average time to results (TTR) was measured at 1:10 of end fluorescence. Error bars represent the standard deviation across six technical replicates. Results show that MDX135 ( blue ) has a shorter TTR than NEB WarmStart ® ( orange ) amplification across all sample types. The data suggests that MDX135 performs better with faster amplification across the range of inhibitors.
70
60
50
40
30
20
10
Lyophilized | NEB WarmStart ®
0
0% inhibitor
30% Saliva
30% UTM
30% VTM
30% Sputum in UTM
30% Sputum
NTC
Superior performance compared to competitors
Higher specificity using samples containing 15% sputum
Lyo-Ready ™ Direct DNA LAMP Saliva (MDX134)
Amplification of Mycoplasma pneumoniae genomic DNA (200 copies) was compared using Lyo-Ready ™ Direct DNA LAMP Saliva (MDX134) against NEB WarmStart ® in samples containing 0% or 15% artificial sputum. Reactions were incubated at 65 °C for 60 minutes, and TTR was measured at 1:10 of end fluorescence. No NTCs were detected in this assay. Lyo-Ready ™ Direct DNA LAMP Saliva (MDX134) with 0% ( light blue ) and 15% ( dark blue ) artificial sputum demonstrated stronger inhibitor tolerance, retaining its fast TTR in the presence of 15% artificial sputum. In contrast, NEB WarmStart ® with 0% artificial sputum ( orange ) amplified slower, as shown by a later TTR, than either reaction using Lyo-Ready ™ Direct DNA LAMP Saliva. In addition, in samples containing 15% artificial sputum ( red ), NEB WarmStart ® had a delayed amplification of 40 min and the longest TTR of all the reactions.
MDX134 0% sputum | MDX134 15% sputum | NEB WarmStart ® 0% sputum | NEB WarmStart ® 15% sputum
Greater assay sensitivity and reproducibility using samples containing up to 25% sputum or 20% saliva Lyo-Ready ™ Direct DNA LAMP Saliva (MDX134)
Lyo-Ready ™ Direct DNA LAMP Saliva (MDX134) ( red ) was lyophilized with Mycoplasma pneumoniae primers and used for the amplification of 2,000, 200 and 20 copies of M. pneumoniae genomic DNA and the results compared to the same primers and DNA for fresh MDX134 liquid mix ( blue ) and NEB WarmStart ® ( orange ), in the presence of 0% and 25% artificial sputum. Reactions were incubated at 65 °C for 60 minutes, and TTR was measured at 1:10 of end fluorescence. Error bars represent the standard deviation across four technical replicates. The results illustrate the increased sensitivity and greater reproducibility of the Lyo-Ready ™ Direct DNA LAMP Saliva, with earlier TTR (faster) and a lower standard deviation.
M. pneumoniae
70 60 50 40 30 20 10 0
2000 cop/rxn
200 cop/rxn No inhibitor
20 cop/rxn
2000 cop/rxn
200 cop/rxn 25% Sputum
20 cop/rxn
Lyo-Ready ™ Direct RNA/DNA LAMP Saliva (MDX135)
Zika Virus
Dengue Type 2 Virus
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
0
2000 ipc
1000 ipc
500 ipc
ntc
2000 ipc
1000 ipc
500 ipc
ntc
2000 ipc
1000 ipc
500 ipc
ntc
2000 ipc
1000 ipc
500 ipc
ntc
No inhibitor
20% Saliva
No inhibitor
20% Saliva
Lyo-Ready ™ Direct RNA/DNA LAMP Saliva (MDX135) ( red ) was lyophilized with Zika virus or Dengue type 2 virus primers and used for the amplification of 2,000, 1 000 and 500 copies of the viral RNA and the results compared to the same primers and viruses for fresh liquid mix ( blue ) and NEB WarmStart ® ( orange ), in the presence of 0% and 20% human saliva. Reactions were incubated at 65 °C for 60 minutes, and TTR was measured at 1:10 of end fluorescence. Error bars represent standard deviation across five technical replicates. The results illustrate the increased sensitivity of the Lyo-Ready ™ Direct RNA/DNA LAMP Saliva, with earlier TTR.
Lyophilized | Liquid | NEB WarmStart ®
Maintains consistent performance in samples containing up to 60% saliva
Lyo-Ready ™ Direct RNA/DNA LAMP Saliva (MDX135)
25
Lyo-Ready ™ Direct RNA/DNA LAMP Saliva (MDX135) was lyophilized with Mycobacterium tuberculosis (MTB) primers and resuspended with increasing amounts of human saliva containing M. tuberculosis (from 10% - 72%). Reactions were incubated at 65 °C for 60 minutes, and TTR was measured at 1:10 of end fluorescence. The results illustrate that Lyo-Ready ™ Direct RNA/DNA LAMP Saliva maintains a robust performance up 60% saliva, and demonstrates consistently late NTCs.
20
15
10
5
0
0% 10%
20%
30%
50%
60%
Saliva (%)
Lyophilized format maintains a stable shelf-life for more than 12 months
Lyo-Ready™ Direct RNA/DNA LAMP Saliva (MDX135)
20
Freshly prepared Lyo-Ready ™ Direct RNA/DNA LAMP mix (MDX135) ( red ) was compared to lyophilized mix that had been incubated for 1 month at 37°C in an accelerated stability study ( blue ). Both mixes were amplified using PMSD4 3T3 mouse cell primers and reactions were incubated at 65 °C for 60 minutes. TTR was measured at 1:10 of end fluorescence and error bars represent the standard deviation across six technical replicates. Results suggest that the lyophilized mix retains full activity following accelerated stability tests with a projected stability of greater than 12 months at ambient temperature.
16
12
8
4
0
Lyo
Wet
Ordering information: USA 5171 Wilfong Road
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