Inhibitor-Tolerant LAMP & RT-LAMP Mixes
Stool-Specific Mixes
Significantly Higher Tolerance to Inhibitors Found in Crude Stool Material
PRODUCT
CAT. NO.
VOLUME REACTIONS
5 mL
800
MDX144 | Lyo-Ready Direct DNA LAMP Stool
Lyo-Ready Direct DNA LAMP Stool Mix
MDX144
50 mL
8,000
Adenovirus
Salmonella typhimurium
10 20 30 40 50 60
10 20 30 40 50 60
5 mL
800
Lyo-Ready Direct RNA/DNA LAMP Stool Mix
MDX145
50 mL 8,000 Diarrhoeal disease is the 8 th leading cause of death globally and the 2 nd leading cause of death in children under the age of five. It is caused by a variety of bacterial, viral and parasitic organisms and is usually spread through the fecal-oral route or by foodborne transmission. Stool has been used a diagnostic specimen to detect the presence of these organisms, and also to diagnose certain cancers and inform of overall gut health. In order to provide the right treatment and prevent serious complications, rapid identification of gastrointestinal infections is crucial. This can be carried out using point-of-care (POC) diagnostic tests that are simple to use, involve minimal-to-nosample pre-processing, do not require cold room storage and are as sensitive and specific as laboratory-based tests. Assays using stool as a specimen can pose a substantial challenge for POC assays as stool samples can be complex, containing food debris, desquamated epithelial cells and mucus, and potential LAMP inhibitors such as bile salts, polysaccharides, hematin and catabolic substances. Meridian has developed Lyo-Ready Direct DNA LAMP Stool and Lyo-Ready Direct RNA/DNA LAMP Stool Mixes to combine high inhibitor tolerance with fast, robust isothermal amplification to enable highly sensitive direct detection using crudely processed stool samples. The mixes contain excipients and an optimized buffer system that is compatible with lyophilization to create ambient-temperature stable assays which have more than two-year’s shelf-life; and are uniquely designed to overcome very high concentrations of inhibitors and so do not require a DNA or RNA purification step to purify a patient sample. Superior Assay Sensitivity in Challenging Environments such as Low Copy Input and High Bile Salt Concentration
0
0
0 mg/mL
6.6 mg/mL
0 mg/mL
6.6 mg/mL
Campylobacter jejuni
Escherichia coli O157
10 20 30 40 50 60
10 20 30 40 50 60
0
0
0 mg/mL
6.6 mg/mL
0 mg/mL
6.6 mg/mL
The impact of stool inhibitors on LAMP performance was tested for Lyo-Ready Direct DNA LAMP Stool ( MDX144 - brown ) in liquid format, NEB WarmStart ® ( green ) and Thermo SuperScript TM IV ( blue ). Mixes were prepared with primers for Salmonella typhimurium , Adenovirus, Campylobacter jejuni or E. coli O157 in the presence of 0 mg/mL or 6.6 mg/mL human stool and genomic DNA from Salmonella , Campy, E. coli or inactivated adenovirus. 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 MDX144 has the shortest TTR even in the presence of 6.6 mg/ mL stool, whereas NEB WarmStart ® and Thermo SuperScript TM IV have significantly delayed TTRs. The results demonstrate that MDX144 has higher tolerance to inhibitors found in stool compared to other commercial mixes. MDX144 | NEB WarmStart ® | Thermo SuperScript TM
MDX145 | Lyo-Ready Direct RNA/DNA LAMP Stool
Rotavirus
RSV virus
Zika virus
10 20 30 40 50 60
10 20 30 40 50 60
10 20 30 40 50 60
A MDX144 | Lyo-Ready Direct DNA LAMP Stool
B MDX145 | Lyo-Ready Direct RNA/DNA LAMP Stool
Salmonella typhimurium
Respiratory Syncytial Virus (mg/mL)
60
60
0
0
0
0 mg/mL
12.5 mg/mL
0 mg/mL
12.5 mg/mL
0 mg/mL
12.5 mg/mL
50
50
Norovirus
Adenovirus
10 20 30 40 50 60
10 20 30 40 50 60
40
40
30
30
20
20
10
10
0
0
0
0
0 mg/mL
12.5 mg/mL
0 mg/mL
12.5 mg/mL
1000
100
10
1
0
3
5
NTC
0
3
5
NTC
0
3
5
NTC
MDX144/MDX145 | NEB WarmStart ® | Thermo SuperScript TM
MDX145 | NEB WarmStart ® | Thermo SuperScript TM
(A) Lyo-Ready Direct DNA LAMP Stool ( MDX144 - brown ) in liquid format was used for the amplification of 1,000, 100, 10 and 1 copies of Salmonella DNA in the presence of 3 mg/mL bile salts and (B) Lyo-Ready Direct RNA/DNA LAMP Stool ( MDX145 - brown ) liquid format was used for the amplification of Respiratory Syncytial Virus (RSV) in the presence of 0 mg/mL, 3 mg/mL and 5 mg/mL bile salts. The results were compared using the same primers and DNA/RNA template for NEB WarmStart ® ( green ) and Thermo SuperScript TM IV ( blue ). 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 demonstrate the high sensitivity of Lyo-Ready Direct DNA LAMP Stool, even at 1 copy of DNA, and both mixes’ high tolerance to bile salt.
The impact of stool inhibitors on LAMP performance was tested for Lyo-Ready Direct RNA/DNA LAMP Stool (MDX145 - brown ) in liquid format, NEB WarmStart ® ( green ) and Thermo SuperScript TM IV ( blue ). Mixes were prepared using inactivated viruses (Rotavirus, Norovirus, RSV, Zika (RNA viruses) and Adenovirus (ADV) (DNA virus)), the corresponding inactivated virus and with 0 mg/mL or 12.5 mg/mL stool. 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 MDX145 has a shorter TTR than NEB WarmStart ® and Thermo SuperScript TM IV in the presence of human stool. MDX145 also demonstrated better performance with faster amplification across all RNA and DNA templates in the presence and absence of human stool.
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