Custom LAMP Primers in Partnership with New England Biolabs

• Lamp Primer Design
• Quality Assurance
• SARS-CoV-2 Targets
• References

Combined with real time detection, LAMP (loop-mediated isothermal amplification) has been a popular isothermal amplification technology used to create COVID-19 molecular diagnostics. Among other reasons, this is likely because LAMP, specifically RT-LAMP (reverse transcription LAMP) in the case of SARS-CoV-2, is a robust method for specific detection of nucleic acids.

Though these methods are robust, it is still best to optimize the reaction as well as its components. This is especially true considering LAMP is well-recognized for producing spurious amplicons that can lead to false positive results¹. While it is up to the assay developer to optimize the reaction, vendors must lead the way optimizing the components.

The strand displacing polymerase, e.g. Bst DNA Polymerase, which catalyzes the LAMP reaction, is an important component that has properties subject to direct improvement, e.g. via site-directed mutagenesis². A quick and indirect way of “improving the polymerase” is by enhancing another equally-important component, the primers. The best way of doing this is by fine-tuning the purification methods used in the manufacturing process (Figure 1). Either Desalt or HPLC purification of LAMP oligonucleotides is common, and it is often possible to modify both methods to create optimized primers.

Custom LAMP Primers, a derivative of our iScale Oligos™, are intended to maximize the function of LAMP primers in assays to be used as life science research tools, molecular diagnostics, and laboratory developed tests.

Figure 1. High-level overview of the main steps used in the manufacturing process for Custom LAMP Primers (steps upstream from Synthesis and downstream from Purification have been omitted for simplicity).

LAMP Primer Design

For background on Custom LAMP Primer design as well as access to a complimentary design tool, please see the excellent resources available from New England Biolabs.
 

Product Benefits

• Amounts available to meet needs from R&D to commercialization
• Consultation with our scientific team to set final specifications
• Specialized purification, ensuring high-performance primers

Product Features

For anything marked as ‘Inquire’ below or If you have needs that are different from the other general specifications presented, please send a request to dnaoligos@sial.com.

Custom LAMP Primers Specifications

*At minimum, we recommend that the FIP/BIP primers be purified via HPLC.

Quantities	• 10 mg to 2 g (inquire for larger quantities)
Purification*	• Desalt (undefined) & HPLC (≥85%)
Sequence Lengths	• 10 to 50 bases (inquire for longer lengths)
Modifications	• Inquire for feasibility of modifications
Quality Control	• 100% mass spectrometry • Analytical HPLC available  • Certificate of Analysis available
Format	• Supplied dry or in solution in tubes (inquire for feasibility of other formats)

Quality Assurance

At the foundation of our manufacturing processes is a robust Quality Management System (QMS), which drives compliance to our following Quality Registrations:

• ISO 9001:2015 for manufacturing research-grade oligonucleotides
• ISO 13485:2016 for manufacturing diagnostic-grade oligonucleotides
• ISO 14001:2015 for effective environmental management

SARS-CoV-2 LAMP Primers

In addition to being a robust method as described above, colorimetric RT-LAMP is rapidly growing in popularity as a molecular diagnostic, especially for SARS-CoV-2, because it allows for rapid detection with minimal instrumentation. Currently, several EUA (Emergency Use Authorization) tests use the primers in Table 1, which are described in the paper, Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride³. Direct testing by several end users has revealed that our Custom LAMP Primers have led to exceptional performance with the E1, N2, and As1e primer sets.

SARS-CoV-2 Targets

E1 Sequence Set
Oligo Name	Sequence	Typical Primer Ratios*	Recommended Purification
E1-F3	TGAGTACGAACTTATGTACTCAT	1X	Desalt
E1-B3	TTCAGATTTTTAACACGAGAGT	1X	Desalt
E1-FIP	ACCACGAAAGCAAGAAAAAGAAGTTCGTTTCGGAAGAGACAG	8-10X	HPLC
E1-BIP	TTGCTAGTTACACTAGCCATCCTTAGGTTTTACAAGACTCACGT	8-10X	HPLC
E1-LF	CGCTATTAACTATTAACG	2X	Desalt
E1-LB	GCGCTTCGATTGTGTGCGT	2X	Desalt

N2 Sequence Set
Oligo Name	Sequence	Typical Primer Ratios*	Recommended Purification
N2-F3	ACCAGGAACTAATCAGACAAG	1X	Desalt
N2-B3	GACTTGATCTTTGAAATTTGGATCT	1X	Desalt
N2-FIP	TTCCGAAGAACGCTGAAGCGGAACTGATTACAAACATTGGCC	8-10X	HPLC
N2-BIP	CGCATTGGCATGGAAGTCACAATTTGATGGCACCTGTGTA	8-10X	HPLC
N2-LF	GGGGGCAAATTGTGCAATTTG	2X	Desalt
N2-LB	CTTCGGGAACGTGGTTGACC	2X	Desalt

Table 1. E1, N2, and As1e primer sets currently used with several EUA tests that detection SARS-CoV-2. Included are typical primer ratios and recommended purifications.

As1e Sequence Set
Oligo Name	Sequence	Typical Primer Ratios*	Recommended Purification
As1e-F3	CGGTGGACAAATTGTCAC	1X	Desalt
As1e-B3	CTTCTCTGGATTTAACACACTT	1X	Desalt
As1e-FIP	TCAGCACACAAAGCCAAAAATTTATTTTTCTGTGCAAAGGAAATTAAGGAG	8-10X	HPLC
As1e-BIP	TATTGGTGGAGCTAAACTTAAAGCCTTTTCTGTACAATCCCTTTGAGTG	8-10X	HPLC
As1e-LF	TTACAAGCTTAAAGAATGTCTGAACACT	2X	Desalt
As1e-LB	TTGAATTTAGGTGAAACATTTGTCACG	2X	Desalt

*Typical primer ratios means that FIP/BIP must be present at 8-10-fold higher than F3/B3, and LF/BF must be present at 2-fold higher for the LAMP chemistry to function properly.

References

1. Jiang YS, Bhadra S, Li B, Wu YR, Milligan JN, Ellington AD. 2015. Robust Strand Exchange Reactions for the Sequence-Specific, Real-Time Detection of Nucleic Acid Amplicons. Anal. Chem.. 87(6):3314-3320. https://doi.org/10.1021/ac504387c

2. Maranhao A, Bhadra S, Paik I, Walker D, Ellington AD. An improved and readily available version of Bst DNA Polymerase for LAMP, and applications to COVID-19 diagnostics. https://doi.org/10.1101/2020.10.02.20203356

3. Zhang Y, Ren G, Buss J, Barry AJ, Patton GC, Tanner NA. 2020. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. BioTechniques. 69(3):178-185. https://doi.org/10.2144/btn-2020-0078

4. Huang X, Tang G, Ismail N, Wang X. 2022. Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva. eBioMedicine. 75103736. https://doi.org/10.1016/j.ebiom.2021.103736

For technical assistance, please consult our technical services group.