Meridian Lyo-Ready Direct LAMP Stool Master Mixes

Lyo-Ready TM Direct LAMP Stool Mixes

Specimen-specific TM mixes designed for fast, direct DNA or RNA isothermal amplification from stool specimens

Meridian’s Lyo-Ready TM Direct DNA LAMP Stool and RNA/DNA LAMP Stool mixes are ready-to-use, 4x concentrated, fully optimized mixes for isothermal amplification methods such as LAMP and designed for rapid, sensitive amplification of RNA or DNA from crudely processed stool specimens. Diarrhoeal disease is the 8th leading cause of death globally and the 2nd 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. Diagnostic tests, especially point-of-care (POC), are essential to enable the rapid identification of gastrointestinal infections and provide the right treatment to prevent serious complications and prevent its spread. Ideally, POC diagnostics are simple to use, involve minimal-to-no-sample 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 PCR inhibitors such as bile salts, polysaccharides, hematin and catabolic substances. Typically, complex samples require pre- processing to extract the DNA or RNA prior to testing. Lyo-Ready Direct DNA LAMP Stool and RNA/DNA LAMP Stool mixes are uniquely designed for POC assays and enable direct analysis straight from crude stool specimens without DNA or RNA extraction. They contain an optimized blend of additives to negate the effects of enzyme inhibitors while maintaining the quality and integrity of nucleic acids found in patient samples. In addition, the mixes are glycerol-free and are suitable to use in a liquid format or they can be lyophilized to create ambient- temperature stable assays which do not require refrigeration. As ready-to-use mixes, no further optimization is required aside from the addition of disease-specific primers and probes. Lyo-Ready Direct DNA LAMP Stool and RNA/DNA LAMP Stool mixes are ideal solutions to simplify the development of LAMP assays for POC and they offer unique advantages including direct detection and the option for down-stream lyophilization.

Comparison studies demonstrate their superior performance in inhibitor tolerance, sensitivity, and reproducibility against other commercially available mixes.

Product Highlights

PRODUCT

CAT NO. VOLUME REACTIONS

• High inhibitor tolerance, suitable for the development of rapid, direct LAMP stool tests • Very sensitive detection, down to single-digit copies of DNA or RNA • Formulated for use as a liquid or lyophilization, ideal for developing ambient-temperate stable POC assays • Validated for the detection of enteric pathogens in crude stool samples

5 mL 800 Rxns 50 mL 8,000 Rxns 5 mL 800 Rxns 50 mL 8,000 Rxns

Lyo-Ready Direct DNA LAMP Stool

MDX144

Lyo-Ready Direct RNA/DNA LAMP Stool

MDX145

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Significantly Higher Tolerance to Inhibitors Found in Crude Stool Material

Lyo-Ready ™ Direct DNA LAMP Stool (MDX144)

The impact of stool inhibitors on LAMP performance was tested for Lyo-Ready ™ Direct DNA LAMP Stool (MDX144) in liquid format ( blue ), NEB WarmStart ® ( orange ) and Thermo SuperScript ™ IV ( black ). 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. 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 6.6mg/ml stool, whereas NEB WarmStart ® and Thermo SuperScript ™ IV have significantly delayed TTRs. The results demonstrate that MDX144 has higher tolerance to inhibitors found in stool compared to other commercial mixes.

Adenovirus

Salmonella typhimurium

60

60

50

50

40

40

30

30

20

20

10

10

0

0

0 mg/mL

6.6 mg/mL

0 mg/mL

6.6 mg/mL

Campylobacter jejuni

Escherichia coli O157

60

60

50

50

40

40

30

30

20

20

10

10

0

0

0 mg/mL

6.6 mg/mL

0 mg/mL

6.6 mg/mL

MDX144 | NEB WarmStart ® | Thermo SuperScript ™ IV

Lyo-Ready ™ Direct RNA/DNA LAMP Stool (MDX145)

Rotavirus

RSV virus

Zika virus

60

60

60

50

50

50

40

40

40

30

30

30

20

20

20

10

10

10

0

0

0

0 mg/mL

12.5 mg/mL

0 mg/mL

12.5 mg/mL

0 mg/mL

12.5 mg/mL

Norovirus

Adenovirus

60

60

50

50

40

40

30

30

20

20

10

10

0

0

0 mg/mL

12.5 mg/mL

0 mg/mL

12.5 mg/mL

MDX145 | NEB WarmStart ® | Thermo SuperScript ™ IV

The impact of stool inhibitors on LAMP performance was tested for Lyo-Ready ™ Direct RNA/DNA LAMP Stool (MDX145) liquid format ( blue ), NEB WarmStart ® ( orange ) and Thermo SuperScript ™ IV ( black ). 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 ™ 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.

Efficient Amplification in Samples Containing a High Concentration of Stool

Lyo-Ready™ Direct RNA/DNA LAMP Stool (MDX145) lyophilized

Lyo-Ready ™ Direct RNA/DNA LAMP Stool (MDX145) was lyophilized and resuspended with increasing amounts of human stool (1.66 mg/mL – 19.98 mg/mL) containing inactivated RSV and primers for the RSV. 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 six technical replicates. The results illustrate that Lyo-Ready ™ Direct RNA/DNA LAMP Stool maintains a robust performance up 19.98 mg/mL stool.

20.00

15.00

10.00

5.00

0.00

0 mg/mL

1.66 mg/mL

3.33 mg/mL

6.66 mg/mL

9.99 mg/mL

13.32 mg/mL

16.65 mg/mL

19.98 mg/mL

Superior Assay Sensitivity in Challenging Environments such as Low Copy Input and High Bile Salt Concentration

A) Lyo-Ready ™ Direct DNA LAMP Stool (MDX144)

B) Lyo-Ready ™ Direct RNA/DNA LAMP Stool (MDX145)

Respiratory Syncytial Virus

Salmonella typhimurium

60

60

50

50

40

40

30

30

20

20

10

10

0

0

1000

100

10

1

MDX144 | NEB WarmStart ® | Thermo SuperScript ™ IV

MDX145 | NEB WarmStart ® | Thermo SuperScript ™ IV

A) Lyo-Ready ™ Direct DNA LAMP Stool (MDX144) liquid format ( blue ), was used for the amplification of 1,000, 100 and 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) liquid format ( blue ) 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 ® ( orange ) and Thermo SuperScript ( black ). 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.

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Faster Time-to-Results and Assay Reproducibility Compared to Other LAMP Mixes

A) Lyo-Ready ™ Direct DNA LAMP Stool (MDX144)

B) Lyo-Ready ™ Direct RNA/DNA LAMP Stool (MDX145)

60

60

40

40

20

20

0

0

1000

100

10

1

2000

1000

500

100

Liquid format | NEB WarmStart ®

A) Lyo-Ready ™ Direct DNA LAMP Stool (MDX144) in liquid format ( blue ) was used in a 10-fold serial dilution of Salmonella DNA (1,000, 100, 10, and 1 copy respectively) and B) Lyo-Ready ™ Direct RNA/DNA LAMP Stool (MDX145) in liquid format ( blue ) was used in a 10-fold serial dilution of RNA (2,000, 1,000, 500 and 100 copies respectively) of Norovirus and the results were compared to NEB WarmStart ® ( orange ) using the same primers and DNA/RNA. 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 LAMP Stool mixes, with earlier TTR (faster) and a lower standard deviation.

Stable Shelf-life for 12+ Months in Lyophilized Format

A) Lyo-Ready ™ Direct DNA LAMP Stool (MDX144)

B) Lyo-Ready ™ Direct RNA/DNA LAMP Stool (MDX145)

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

5

0

0

0 mg/mL

3.3 mg/mL

0 mg/mL

3.3 mg/mL

Freshly prepared mix | Lyophilized mix

The performance of freshly prepared Lyo-Ready™ Direct DNA LAMP Stool (MDX144, blue ) and Lyo-Ready™ Direct RNA/DNA LAMP Stool mix (MDX145, blue ) were compared to lyophilized versions of the mixes that had been incubated for 1 month at 37°C in an accelerated stability study ( red ). Amplification reactions were carried out at 65 °C for 60 minutes using (A) MDX144, Adenovirus primers, inactivated adenovirus and either 0 mg/mL or 3.3 mg/mL human stool or (B) MDX145, Norovirus primers, inactivated norovirus and either 0 mg/mL or 3.3 mg/mL human stool. Error bars represent the standard deviation across four technical replicates. The results indicate that the lyophilized mixes have a projected stability of at least 12 months at ambient temperature.

Ordering information: USA 5171 Wilfong Road Memphis, Tennessee 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223

Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.meridianbioscience.com/lifescience

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