03/​27
2020

6 new Articles, referring to the use of CRISPR/​Cas and HybriDetect

DNA and RNA Detection with the Help of HybriDetect

New techniques have been developed for the specific detection of DNA or RNA sequences. They all have in common, that they use CRISPR/​Cas12 or Cas13. The enzyme recog­nizes a specific target sequence. After binding to this sequence, its endonu­clease activity is activated and it cleaves any reporter sequence. This technique can be easily combined with our Lateral Flow Assay HybriDetect for the visual­ization of the results.

03/​20
2020

HybriDetect and Temper­ature Effects

Using higher Temper­a­tures: Part of the NAFLIA Toolbox

Higher Temper­a­tures: Stability aspects and assay Parameters

The general robustness is a charac­ter­istic feature of lateral flow devices (LFD’s). Even during longer trans­ports and extreme weather condi­tions LFD’s remain stable. Temper­ature is an important factor when it comes to robustness. Therefore our HybriDetect test strips are regularly tested at higher temper­a­tures.

Especially in the devel­opment of NALFIA’s (Nucleic Acid Lateral Flow Immunoassays), the temper­ature can influence the test result signif­i­cantly. Higher temper­a­tures can also be under­stood as tool in the assay procedure to achieve certain results. The hybridization of amplifi­cates is classified as partic­u­larly inter­esting. The use of a defined temper­ature is a crucial parameter to maintain specific hybridizing condi­tions.

Higher Temper­a­tures: Perfor­mance Test

For this reason, Milenia Biotec GmbH took a closer look at this issue: The universal test strips HybriDetect (MGHD1) and HybriDetect 2T (MGHD2 1) were examined under different condi­tions with dilution series of labelled dsDNA. The purified labelled amplifi­cates were placed into running buffer and warmed to the desired temper­ature. Test strips were prewarmed as well for 30 minutes. After rewarming Lateral Flow Analysis was initiated. After five minutes signals were documented. The overall results are shown in the following figure.

Figure: Influence of higher temper­a­tures to LFA-​​performance of HybriDetect test strips. Red triangles indicate temper­ature related nonspe­cific signals on test lines.

The results illus­trate the robustness of the dipsticks. Even at 65 °C, a sensitive analysis is possible without a visible loss of perfor­mance. Nonspe­cific signal appear over 70°C, especially on the testline 2 of the HybriDetect 2T (red triangles in the figure). This effect is easy to explain. Like most LFD’s the HybriDetect platform includes antibodies. If these antibodies are exposed to temper­a­tures over 70°C, denat­u­ration takes place. This results in non-​​specific signals.

However, the full function­ality of the Hybrid­Detect test strips was demon­strated in the tested range of 20°C to 65 °C. Temper­ature should be considered as an additional tool in assay devel­opment. Try to profit from this feature!

03/​13
2020

Is it possible to develop a nucleic acid based COVID-​​19 test with detection of the amplicons via the lateral flow test “Milenia HybriDetect”?

Recently Milenia Biotec GmbH received several customer inquiries related to COVID-​​19 detection, due to the fact, that HybriDetect may be used for the detection of RPA, LAMP or PCR amplicons. Following up these inquiries, we a liter­ature search on the SHERLOCK (Specific High Sensi­tivity Enzymatic Reporter UnLOCKing) technique, which is a powerful tool to detect genetic infor­mation coming from viruses using CRISPR.

What is HybriDetect?

The HybriDetect is a lateral flow dipstick (LFD), which is capable to detect different molecules, including gene ampli­fi­cation products, proteins and antibodies. A commonly used appli­cation for our test is the detection of gene ampli­fi­cation products resulting from PCR, LAMP or RPA. The under­lying genetic infor­mation can be detected regardless which kind of organism need to be detected.Finally, labeled primers must be intro­duced in the ampli­fi­cation step, so that the resulting fragments are labeled and can be detected by the HybriDetect dipstick.

Results can be reported within 5 minutes after lines become visible on the test Strip.

COVID-​​19 Detection Methods

At the moment, the standard method for detection of COVID-​​19 is a qRT-​​PCR. Obviously, there are several reasons why qRT-​​PCR cannot be used as a POCT. First of all, it takes several hours to do a qPCR, second you need highly trained personnel and third special equipment is essential. In the current situation, with the entire world dealing with the COVID-​​19 outbreak, it is of great interest to develop rapid tests for POC testing of the new corona virus to help people in sparsely populated areas where labora­tories are difficult to reach.

 

Here we give a short overview of the latest publi­ca­tions dealing with the rapid detection of COVID-​​19 combined with the HybriDetect Lateral Flow Method for visual­ization.

DETECTR

Broughton et al. already published a paper, how to detect SARS_​CoV-​​2 using LAMP, Cas12 and our HybriDetect within 30 minutes. They are using isothermal pre-​​amplification with primers published by the WHO and CDC. As the authors mentioned in the paper, this appli­cation would be a helpful tool for POCT testing in emergency depart­ments, airports etc..

For more infor­mation see the original paper:

https://mammoth.bio/2020/02/15/white-paper-a-protocol-for-rapid-detection-of-sars-cov-2-using-crispr-sars-cov-2-detectr/

SHERLOCK

The group of the inventor of the SHERLOCK method Feng Zhang also described a method for COVID-​​19 detection using CRISPR. The scien­tists were able to detect synthetic COVID-​​19 virus RNA fragments between 20 and 200 aM (10 – 100 copies per µl of input). They used purified RNA as Input for an RT-​​RPA before the Cas13 assay. The whole method takes less than one hour.

The following picture shows an example of the COVID-​​19 detection using our HybriDetect.

For more infor­mation see the original paper:

https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf

Zhang et al give a precise description, how the SHERLOCK technique is done in nature protocol:

http://dx.doi.org/10.1038/s41596-0190210-2

Comparison of the three Methods (as described by Broughton et al.)

Here you can find a video from the McGovern Institute describing the SHERLOCK technique in combi­nation with the HybriDetect in General