Welcome!

Amplex Diagnostics are known for their LAMP technology (utilised in the eazyMini and eazyPlex test range) and applications in fast, accurate and reliable solutions in as little as 20 minutes for detection of a whole range of common, and uncommon, pathogens.

They have now innovatively combined LAMP technology with CRISPR to produce their new products eazySNP (s.ingle n.ucleotide p.olymorphs), with their first application being used to target one of the world's most significant multi-drug-resistant yeast pathogens: Candidozyma auris.

eazySNP C. auris FKS1 S639 is the first CE-IVD-certified diagnostic test of its kind, and enables the highly specific detection of clinically relevant mutations in the FKS1 gene associated with echinocandin resistance.

Echniocandins currently represent the first-line treatment option for C. auris infections, meaning the rapid identification of resistance-associated variants is of considerable clinical importance.

The assay is performed directly from a yeast colony, using the eazyMini platform, and delivers results within just 45 minutes. This means that the eazySNP technology therefore provides, for the first time, a rapid targeted molecular solution for the detection of this important resistance marker.

The technology: combining CRISPR & LAMP in one test

• Using an optimised LAMP-primerset the target region of the gene of interest (carrying the position of SNP in a non-binding region between the FIP- and BIP-primers) is amplified.
• With the help of a CRISPR RNA (crRNA), the type-III CRISPR-Cas protein complex detects the sequence of interest (SNP or insertion) with high specificity. 
• Upon recognition and binding, the type-III CRISPR-Cas complex produces cyclic oligoadenylate (cOA) from adenosine triphosphate (ATP) molecules. The generated cOA subsequently activates an RNase, an enzyme that cleaves a fluorescent reporter RNA. 
• After cleavage of the fluorescent reporter, the resulting fluorescent signal can be measured. 

How does LAMP work?

If you've never heard of LAMP technology before, think of it like PCR, but not PCR. Here's a handy graphic explaining the 9 steps of the LAMP reaction. 

LAMP has many benefits over PCR, such as no extraction step, and much faster run-times. If you would like more information regarding LAMP and it's use in Amplex Diagnostic's eazyPlex tests, please reach out to us at: welcome@bioconnections.co.uk/ 01782 516010. 

The 9 Step of the LAMP reaction: 

Written here if you struggle reading the above graphic 

1. Initiation by FIP: The FIP anneals to the template DNA and initiates DNA synthesis using DNA polymerase with strand displacement activity, releasing a single-stranded DNA. 
2. Elongation from F2 region: Through the activity of DNA polymerase with strand displacement activity, a DNA strand complementary to the template DNA is synthesised, starting from the 3' end of the F2 region of the FIP. 
3. Initiation by F3 primer: The F3 primer anneals to the F3c region, outside of FIP, on the target DNA and initiates strand displacement DNA synthesis, releasing the FIP-linked complementary strand. 
4. Double strand formation: A double strand is formed from the DNA strand synthesised from the F3 primer and the template DNA strand. 
5. Release of single strand: The FIP-linked complementary strand is released as a single strand because of the displacement by the DNA strand synthesised from the F3 primer. Then, this released single strand forms a stem-loop structure at the 5' end because of the complementary F1c and F1 regions. 
6. BIP-initiated synthesis: This single-strand DNA in Step (5) serves as a template for the BIP-initiated DNA synthesis and subsequent B3-primed strand displacement DNA synthesis. 
7. Double stranded DNA produced: Double-stranded DNA is produced through the processed described in Step (6).
8. Stem-loop (dumbbell) structure: The BIP-linked complementary strand displaced in step (6) forms a structure with stem-loops at each end, which resembles a dumbbell structure. This structure serves as the starting structure for the amplification cycle in the LAMP method (LAMP cycling). 
9. LAMP cycling (amplification): LAMP cycling can be understood as producing the starting structure for LAMP cycling. 

What is CRISPR?

C.lustered R.egularly I.nterspaced S.hort P.alindromic R.epeats

When bacteria and archaea are first infected by a virus, they have a mechanism that causes short fragments of that viral DNA to become integrated into their genomes. These serve as "vaccinations" in the sense that they become the templates for producing small noncoding RNAs known as crRNAs (CRISPR RNAs) that will thereafter destroy the virus, should it reinfect the descendants of the original cell. This aspect of the CRISPR system is similar in principle to adaptive immunity in mammals, in that the cell carries a record of past exposures that is used to protect against future exposures.

The second distinguishing feature of the CRISPR system is that these crRNAs then become associated with special proteins that then allow them to seek out and destroy double-stranded DNA molecules, rather than single-stranded RNA molecules.

Many details of CRISPR-mediated immunity remain to be discovered, however the general process can be outlined in three steps:

1. A short viral DNA sequence is integrated into the CRISPR locus, a CRISPR locus, in its simplest form, consisting of several hundred repeats of a host DNA sequence interspersed with a large collection of sequences (typically 25-70 nucleotide pairs each) that has been derived from prior exposures to viruses and other foreign DNA.
2. RNA is transcribed from the CRISPR locus, processed, and bound to a Cas protein
3. Small crRNA in a complex with Cas seeks out and destroys viral sequences

How the Bacterial CRISPR system has been adapted to edit genomes in a wide variety of species

The CRISPR system uses a guide RNA sequence to target (through complementary base-pairing) double-stranded DNA, which it then cleaves. The gene coding this key component of this system, the bacterial cas9 protein, has been transferred into a variety of organisms, where it greatly simplifies the process of making transgenic organisms.

The CRISPR system has a variety of uses, with its particular power lying with its ability to target cas9 to thousands of different positions across a genome through the simple rules of complementary base-pairing. Thus, if a catalytically inactive Cas9 protein is fused to a transcription activator or repressor, it is possible, in principle, to turn any gene on or off.

Information taken from: Alberts, B. et al. (2022) Molecular Biology of the cell. New York, NY: W.W. Norton & Company. https://chemistry.com.pk/books/molecular-biology-of-the-cell-6e/

Tying it all together

A single eazySNP test strip contains, in each filled tube, six oligonucleotide primers, and DNA polymerase, providing the means for simultaneous, specific amplification of different genes in a single isothermal amplification reaction.

In the presence of relevant DNA sequences, amplicons of the specific target genes are generated. These are detected by a fluorescence measurement of a fluorescent dye that is bound to double-stranded DNA, or are bound by an additionally present, specific type III CRISPR-Cas complex and made detectable by a fluorescence measurement of a cleaved fluorescence reporter.

Therefore, positive signals indicate the specific amplification during the isothermal reaction and therefore the presence of the target gene(s) in the sample under investigation.

If you would like to see what else Amplex Diagnostics has to offer, we have a webpage with all of the information about their entire range! 

If you have any questions regarding Amplex and their eazySNP test, please don't hesitate to reach out to us, we're always happy to have a chat about products! :)

More information? How can we help?

☎️ Telephone: 01782 516010

📧 Email: welcome@bioconnections.co.uk

Thank you for your interest. 🙇