Saturday, December 22, 2018

“0D” Microarrays Of Live Bacteria Using AC Electrokinetics On A Chip

Bacteria have been around for over a billion years and have developed remarkable capabilities to not only cause deadly infections but also to survive adverse environmental conditions (1, 2). This has led to a rich cellular diaspora with diverse geno- and phenotypic variability that is important to characterize owing to its dire implications on mankind.

Researchers are constantly evolving strategies that allow them to study complex cellular processes like cell-cell interactions, cellular metabolic activity, gene function, response to external stimuli (3-7), host-pathogen interactions, and surface-associated redox activities (8) at a single cell level. The foremost requirements of these single cell test platforms are to distinguish, isolate, manipulate, immobilize, and characterize cells in an easy and reproducible manner.

The conventional methods widely used for single cell isolation include fluorescence- or magnetic-activated cell sorting (FACS/MACS), micromanipulation, microdissection, and manual cell picking. While FACS/MACS need high cellular loads, large sample volumes and specific treatment of cells prior to analysis, the remaining techniques have quite a low throughput and require highly-skilled labor for carrying out the experiments. Other methods for cell capture include ink-jet printing on molecularly-patterned templates (9) or dip-pen nanolithography (10), but they again, suffer from poor reproducibility, high cost, and our inability to reuse the substrate.

With recent advances in the field of microfluidics, manipulation and separation of cells on the basis of their dielectric properties, using external electric fields has garnered significant attention. This approach offers numerous advantages such as a high extent of miniaturization, low sample volume, controlled fluid dynamics, ease of sample handling, high throughput, great precision, automation, and label-free operation.

Motivated by these factors, we exploited an electrokinetic phenomenon called alternating current (AC) dielectrophoresis to collect live bacterial cells into grid-like patterns, with each node containing a single cell (see image below). This electric field-assisted assembly not only provided high spatio-temporal control over several thousands of cells simultaneously, but the dynamically-formed cellular arrays were also fully reversible meaning that both the cells and the chips could be retrieved at the end of each capture cycle. This study was recently published in Biosensors and Bioelectronics (111, 2018, 159–165) and showed that model Gram-negative bacteria S.typhi and E.coli can be trapped inside micropatterned conductive circular well electrodes by applying AC electric fields (5 MHz, 5-20 Vpp) across a few microliters of cell suspension.

Dynamic electropatterning of live bacteria into single microwell arrays using AC electrokinetics. The bacteria were fluorescently tagged to allow visualization. Image courtesy Meenal Goel & Shalini Gupta
Shrinking the size of the individual electrodes down to 5 μm allowed single cells to be captured per well with 90% efficiency. This trapping of cells took place due to positive dielectrophoresis that attracted them toward regions of high electric field intensity (microwells in our case). The cells remained alive during the one-hour-long operation, and the overall collection efficiency (from bulk to the surface) was also found to be around 90%.

The potential biotechnological application of our chip was demonstrated in two ways. First, the two cell types were mixed in suspension and collected together using the chip. Counting their relative concentration in the 2D matrix using their phenotypic traits allowed us to directly estimate cellular bulk concentrations in the mixture without generating any calibration curves. Both the total time taken and the volume of the sample used was a fraction of the conventional methods. In the second case, the chip was integrated with an impedance spectroscope to carry out rapid cell viability testing. The response of dielectrophoretically-trapped bacteria against a known antimicrobial peptide was recorded at different drug concentrations and found to be ultrasensitive, reducing the overall time for drug susceptibility testing to just under an hour. This is a significant reduction over standard clinical methods that take several hours to produce outcomes.

In the future, our technology could be used for a wide range of applications including multiplexed sensing, rapid single bacterial profiling in heterogeneous populations, and ultrafast drug susceptibility testing to reduce the use of broad-spectrum antibiotics and, hence, the emergence of new antibiotic-resistant strains.

These findings are described in the article entitled Electric-field driven assembly of live bacterial cell microarrays for rapid phenotypic assessment and cell viability testing, recently published in the journal Biosensors and Bioelectronics. This work was conducted by Meenal Goel, Abhishek Verma, and Shalini Gupta from the Indian Institute of Technology Delhi.

Source: Science Trends

References:

  1. Turner, N. A., Harris, J., Russell, A. D., Lloyd. D. 2000. J. Appl. Microbiol. 89, 751–759.
  2. Davey, M. E., O’Toole, G. A., 2000. Microbiol. Mol. Biol. Rev. 64, 847–867.
  3. Flaim, C.J., Chien, S., Bhatia, S.N., 2005. Nat. Methods 2, 119–125.
  4. Hung, P.J., Lee, P.J., Sabounchi, P., Lin, R., Lee, L.P., 2005. Biotechnol. Bioeng. 89, 1–8.
  5. Lee, M.Y., Dordick, J.S., 2006. Curr. Opin. Biotechnol. 17, 619-27.
  6. van der Meer, J.R., Belkin, S. 2010. Nat. Rev. Microbiol. 8, 511–522.
  7. Ziauddin, J., Sabatini, D.M., 2001. Nature 411, 107–10.
  8. Potma, E. O., de Boeij, W. P., van Haastert, P. J. M., Wiersma., D. A. 2001. Proc. Natl. Acad. Sci. 98, 1577–1582.
  9. Xu, Luping, Robert, Lydia, Ouyang, Qi, Taddei, François, Chen, Yong, Lindner, Ariel B., Baigl, Damien, 2007. Nano Lett. 7, 2068–2072.
  10. Kim, J., Shin, Y.-H., Yun, S.-H., Choi, D.-S., Nam, J.-H., Kim, S.R., Moon, S.-K., Chung, B.H., Lee, J.-H., Kim, J.-H., Kim, K.-Y., Kim, K.-M., Lim, J.-H., 2012. J. Am. Chem. Soc. 134, 16500–16503.

Thursday, October 4, 2018

Glow-in-the-Dark Paper as a Rapid Test for Infectious Diseases

Researchers from Eindhoven University of Technology (The Netherlands) and Keio University (Japan) present a practicable and reliable way to test for infectious diseases: All you need are a special glowing paper strip, a drop of blood and a digital camera, as they write in the scientific journal Angewandte Chemie. Not only does this make the technology very cheap and fast -- after twenty minutes it is clear whether there is an infection -- it also makes expensive and time-consuming laboratory measurements in the hospital unnecessary. In addition, the test has a lot of potential in developing countries for the easy testing of tropical diseases.

The image above shows research leader Maarten Merkx with one copy of the 'glow-in-the-dark' test. Photo: Bart van Overbeeke.

The test shows the presence of infectious diseases by searching for certain antibodies in the blood that your body makes in response to, for example, viruses and bacteria. The development of handy tests for the detection of antibodies is in the spotlight as a practicable and quick alternative to expensive, time-consuming laboratory measurements in hospitals. Doctors are also increasingly using antibodies as medicines, for example in the case of cancer or rheumatism. So this simple test is also suitable for regularly monitoring the dose of such medicines to be able to take corrective measures in good time.

Paper gives light

The use of the paper strip developed by the Dutch and Japanese researchers is a piece of cake. Apply a drop of blood to the appropriate place on the paper, wait twenty minutes and turn it over. "A biochemical reaction causes the underside of paper to emit blue-green light," says Eindhoven University of Technology professor and research leader Maarten Merkx. "The bluer the color, the higher the concentration of antibodies." A digital camera, for example from a mobile phone, is sufficient to determine the exact color and thus the result.

This paper strip (extremely zoomed in) contains two copies of the test. The three glowing dots per test indicate that you can check on three different antibodies within one test. Credit: Bart van Overbeeke.

Sensor protein

The color is created thanks to the secret ingredient of the paper strip: a so-called luminous sensor protein developed at TU/e. After a droplet of blood comes onto the paper, this protein triggers a reaction in which blue light is produced (known as bioluminescence). An enzyme that also illuminates fireflies and certain fish, for example, plays a role in this. In a second step, the blue light is converted into green light. But here comes the clue: if an antibody binds to the sensor protein, it blocks the second step. A lot of green means few antibodies and, vice versa, less green means more antibodies.

Market launched within a few years

The ratio of blue and green light can be used to derive the concentration of antibodies. "So not only do you know whether the antibody is in the blood, but also how much," says Merkx. By measuring the ratio precisely, they suffer less from problems that other biosensors often have, such as the signal becoming weaker over time. In their prototype, they successfully tested three antibodies simultaneously, for HIV, flu and dengue fever. Merkx expects the test to be commercially available within a few years.

Now the production of the paper strips is still handicraft. Photo: Bart van Overbeeke.

Journal Reference:

Keisuke Tenda, Benice van Gerven, Remco Arts, Yuki Hiruta, Maarten Merkx, Daniel Citterio. Paper-Based Antibody Detection Devices Using Bioluminescent BRET-Switching Sensor Proteins. Angewandte Chemie International Edition, 2018

Abstract:

This work reports on fully integrated “sample‐in‐signal‐out” microfluidic paper‐based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET‐based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume‐independent and fully reagent‐free operation, including on‐device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti‐HIV1, anti‐HA, and anti‐DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user‐friendly point‐of‐care testing in low‐resource environments.

Source: Eindhoven University of Technology

Thursday, July 12, 2018

FDA Promotes Rapid Sterility Testing for Human Gene Therapy Products in its Draft Guidance for Investigational New Drug Applications (INDs)

This week, FDA’s Center for Biologics Evaluation and Research (CBER) published six draft guidances relating to gene therapy, three of which cover products for specific disease categories (hemophilia, rare diseases, and retinal disorders), and three of which address manufacturing and clinical study design issues related to gene therapy: chemistry, manufacturing and control (CMC) information in INDs, long term follow-up study design, and testing of retroviral vector-based products.

In a press release, FDA Commissioner Scott Gottlieb, M.D., highlighted “rapid advancements” and “great promise” in the gene therapy space, saying the guidances “are aimed at fostering developments in this innovative field.”  Dr. Gottlieb acknowledged that for some gene therapies, FDA “may need to accept some level of uncertainty” at the time of approval regarding questions related to durability of response, as well as product manufacturing and quality.  He acknowledged the need, however, to assure patient safety and to assure that potential risks are adequately characterized and benefits are adequately demonstrated.

The draft guidance is titled, "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs). Draft Guidance for Industry. July 2018. Clicking on the link will bring up a complete PDF of the guidance document.

The purpose of the draft guidance is to inform sponsors how to provide sufficient CMC information required to assure product safety, identity, quality, purity, and strength (including potency) of the human gene therapy investigational product.

When finalized, the draft guidance will supersede the document entitled “Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs),” dated April 2008.

Because the draft guidance recommends the use of rapid microbiological methods (RMM) for in-process and finished product sterility testing, it is important to understand FDA's position in this regard. As such, I will highlight specific sections in the draft guidance and where appropriate, provide additional interpretation and comment.

Introduction

Human gene therapy products are defined as all products that mediate their effects by transcription or translation of transferred genetic material or by specifically altering host (human)genetic sequences. Some examples of gene therapy products include nucleic acids, genetically modified microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, and ex vivo genetically modified human cells. Gene therapy products meet the definition of “biological product” in section 351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262(i)) when such products are applicable to the prevention, treatment, or cure of a disease or condition of human beings.

The FDA requires all sponsors of investigational new drug products (DPs), including investigational gene therapy products, to describe the CMC information for the drug substance (DS). FDA may place the IND on clinical hold if the IND does not contain sufficient CMC information to assess the risks to subjects in the proposed studies. The CMC information submitted in an IND is a commitment to perform manufacturing and testing of the investigational product, as stated. However, FDA acknowledges that manufacturing changes may be necessary as product development proceeds, and sponsors should submit information amendments to supplement the initial information submitted for the CMC processes. The CMC information submitted in the original IND for a Phase 1 study may be limited, and therefore, the effect of manufacturing changes, even minor changes, on product safety and quality may not be known. Thus, if a manufacturing change could affect product safety, identity, quality, purity, potency, or stability, sponsors should submit the manufacturing change prior to implementation.

What is Required in the IND Application

The IND should include specifications with established acceptance criteria for safety testing at Phase 1. Safety testing includes tests to ensure freedom from extraneous material, adventitious agents, microbial contamination, and replication competent virus. Information on some common safety test methods is provided in more detail in section 3.2.S.4.2, Analytical Procedures.

To maximize the sensitivity of safety testing, it is important that sponsors perform each test at the stage of production at which contamination is most likely to be detected. For example, tests for mycoplasma or adventitious viruses (in vivo or in vitro) should be performed on cell culture harvest material (cells and supernatant) prior to further processing, e.g., prior to clarification, filtration, purification, and inactivation.

Additional testing will depend on the type of gene therapy product and the phase of clinical development. These tests may include assays to assess product characteristics, such as identity, purity (including endotoxin and contaminants, such as residual host cell DNA, bovine serum albumin (BSA), DNase), and potency/strength.

Sponsors should provide a description of all the analytical procedures used during manufacturing to assess the manufacturing process and product quality. In the original IND submission, descriptions should have sufficient detail so that FDA can understand and evaluate the adequacy of the procedures. FDA recommends that sponsors develop detailed SOPs for how analytical procedures are conducted at early stages of product development as a part of the sponsor's quality system. FDA acknowledges that, during product development, analytical methods may be modified to improve control and suitability. However, assay control is necessary during all phases of clinical development to ensure product quality and safety and to allow for comparability studies, following manufacturing changes.

Safety testing on the DS should include microbiological testing, such as bioburden (or sterility, as appropriate), mycoplasma, and adventitious viral agent testing, to ensure product quality. Guidelines and/or procedures for many safety tests have been described in detail, elsewhere (e.g., bioburden [1] sterility [2] mycoplasma [3], adventitious agent testing, and tests for specific pathogens [4]).

Sponsors should list DP specifications in the original IND submission. The testing plan should be adequate to describe the physical, chemical, or biological characteristics of the DP necessary to ensure that the DP meets acceptable limits for identity, strength (potency), quality, and purity. Product lots that fail to meet specifications should not be used in clinical investigation without FDA approval. For early phase clinical studies, FDA recommends that assays be in place to assess safety (which includes tests to ensure freedom from extraneous material, adventitious agents, and microbial contamination) and dose (e.g., vector genomes, vector particles, or genetically modified cells) of the product.

Sponsors should describe the analytical procedures used for testing the DP. If the analytical procedures are the same as those for the DS, sponsors do not need to repeat this information unless there is a matrix effect from the DP on assay performance.

Product Release Testing

FDA recommends that product release assays be performed at the manufacturing step at which they are necessary and appropriate. For example, mycoplasma and adventitious agents release testing is recommended on cell culture harvest material. In addition, sterility, endotoxin, and identity testing are recommended on the final container product to ensure absence of microbial contamination or to detect product mix-ups that might have occurred during the final DP manufacturing steps (e.g., buffer exchange, dilution, or finish and fill steps).

If a DP is frozen before use, FDA recommends that sponsors perform sterility testing on the product prior to cryopreservation so that results will be available before the product is administered to a patient. However, if the product undergoes manipulation after thawing (e.g., washing, culturing), particularly if procedures are performed in an open system, sterility testing may need to be repeated.

FDA also recommends that the results of in-process sterility testing be incorporated into the acceptance criteria for final product specifications.

Alternative Microbiological Methods and Rapid Sterility Tests

Analytical procedures different than those outlined in the United States Pharmacopeia (USP), FDA guidance, or Code of Federal Regulations (CFR) may be acceptable under IND if sponsors provide adequate information on test specificity, sensitivity, and robustness. Examples of alternative methods, which may be needed for live cells, include rapid sterility tests, rapid mycoplasma tests (including PCR-based tests), and rapid endotoxin tests. FDA recommends that sponsors plan to demonstrate equal or greater assurance of the test methodology, compared to a compendial method, prior to licensure, as required under 21 CFR 610.9.

For reference, Sec. 610.9, Equivalent methods and processes, states the following:
Modification of any particular test method or manufacturing process or the conditions under which it is conducted as required in this part or in the additional standards for specific biological products in parts 620 through 680 of this chapter shall be permitted only under the following conditions:
(a) The applicant presents evidence, in the form of a license application, or a supplement to the application submitted in accordance with 601.12(b) or (c), demonstrating that the modification will provide assurances of the safety, purity, potency, and effectiveness of the biological product equal to or greater than the assurances provided by the method or process specified in the general standards or additional standards for the biological product; and
(b) Approval of the modification is received in writing from the Director, Center for Biologics Evaluation and Research or the Director, Center for Drug Evaluation and Research.
FDA recognizes that the compendial sterility test may not be suitable for all products. For example, rapid sterility tests may be needed for ex vivo genetically modified cells administered fresh or with limited hold time between final formulation and patient administration.

For ex vivo genetically modified cells that are administered immediately after manufacturing, in-process sterility testing on sample taken 48 to 72 hours prior to final harvest is recommended for product release. For such products, aside from an in-process sterility test, we also recommend that sponsors perform a rapid microbial detection test, such as a Gram stain, on the final formulated product and a sterility test, compliant with 21 CFR 610.12, on the final formulated product.

Under this approach, the release criteria for sterility would be based on a negative result of the Gram stain and a no-growth result from the 48 to 72 hour in-process sterility test. Although the results of the sterility culture performed on the final product will not be available for product release, this testing will provide useful data. A negative result will provide assurance that an aseptic technique was maintained. A positive result will provide information for the medical management of the subject and trigger an investigation of the cause of the sterility failure. The sterility culture on the final formulated product should be continued for the full duration (usually 14 days) to obtain the final sterility test result, even after the product has been administered to the patient.

In all cases where product release is prior to obtaining results from a full 14-day sterility test, the investigational plan should address the actions to be taken in the event that the 14-day sterility test is determined to be positive after the product is administered to a subject. Sponsors should report the sterility failure to both the clinical investigator and FDA. FDA recommends that sponsors include results of the investigation of cause and any corrective actions in an information amendment submitted to the IND within 30 calendar days after initial receipt of the positive culture test result.

In addition, be aware that a product may sometimes interfere with the results of sterility testing. For example, a product component or manufacturing impurities (e.g., antibiotics) may have mycotoxic or anti-bacterial properties. Therefore, FDA recommends that sponsors assess the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP <71>, Sterility Tests.

DISCUSSION

The draft guidance is a welcome indication that the FDA accepts and encourages the use of alternative and rapid microbiological methods, specifically for sterility testing of short-lived products, such as advanced therapy medicinal products (ATMP; gene and cell therapy). It is appropriate that the guidance document aligns fairly well with 21 CFR 610.12; however, there are some areas that require further clarification.

For example, the use of the 19th Century Gram Stain should be viewed only as a measure of gross contamination because low levels of microbial contaminants would never be observed from a loopful of finished product.

Next, a finished product sterility test that is compliant with 21 CFR 610.12 does not require a 14 day incubation via the compendial USP Sterility Test.

In fact, 21 CFR 610.12 states the following:
Advances in technology in recent years have allowed the development of new sterility test methods that yield accurate and reliable test results in less time and with less operator intervention than the currently prescribed methods. Some examples of novel methods include the Adenosine Triphosphate (ATP) bioluminescence, chemiluminescence, and carbon dioxide head space measurement. Manufacturers may benefit from using such sterility test methods with rapid and advanced detection capabilities. Accordingly, we have amended § 610.12 to promote improvement and innovation in the development of sterility test methods, to address the challenges of novel products that may be introduced to the market in the future, and to potentially enhance sterility testing of currently approved products. This final rule provides manufacturers the flexibility to take advantage of methods as they become available, provided that these methods meet certain criteria.
Because some of these new technologies do not reply on microbial growth, or more raid growth-based methods may be validated to provide an equivalent sterility test result in less than 14 days, the full USP <71> incubation period may not be necessary.  

The teachings in 21 CFR 610.12 do allow for the sterility testing of material other than the finished drug product in its final container. The May 3, 2012 Federal Register Vol. 77, No. 86, Page 26162 – 26175 provides additional guidance on what alternative material may be comprised of, e.g., bulk material or active pharmaceutical ingredient (API), in- process material, stock concentrate material), as appropriate, and as approved in the biologics license application (BLA) or BLA supplement.

Additional guidance was provided in the same CFR reference (see page 26165 in the CFR):
As discussed in the preamble to the proposed rule (76 FR 36019 at 36021), certain allergenic and cell and gene therapy products may need to be tested for sterility at an in- process stage or some other stage of the manufacturing process (e.g., intermediate, API, bulk drug substance) instead of the final container material because the final container material may interfere with the sterility test. Likewise, as discussed in the preamble to the proposed rule, some cell therapy products and cell-based gene therapy products may need to be tested for sterility at an in-process stage or some other stage of manufacturing process because low production volumes may result in an insufficient final container material sample for sterility testing or a short product shelf-life may necessitate administration of the final product to a patient before sterility test results on the final container material are available.
Therefore, based on 21 CFR 610.12, it may be appropriate to test an alternative material and not necessarily the finished product, as long as this strategy is justified and acceptable to FDA. For these reasons, the draft guidance should be clearer on whether a sterility test on the final formulated product is a recommendation or an expectation.

Furthermore, the statement regarding demonstrating the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP <71>, should be extended to alternative or rapid sterility tests. These studies are, in fact, an expected part of the qualification of the alternative method and is usually conducted during the Method Suitability phase of the validation process.

Overall, the draft guidance is further assurance from the FDA that rapid and alternative methods are encouraged for use, especially on product that is short-lived or needs to be administered prior to obtaining results from the compendial sterility test. Although the document is specifically focused on gene therapy products in support of IND applications, the recommendations can be used for the routine release of the same or similar ATMPs.

Training on Rapid Sterility Testing of Gene and Cell Therapy Products

I will be speaking on this same topic during the PDA Europe Conference on Pharmaceutical Microbiology, which will be held in Berlin, Germany on October 15-16. More importantly, I will provide a comprehensive overview of rapid methods, including validation strategies, technology reviews, regulatory acceptance and applications during a two-day training course immediately following the conference on October 17-18. For more information, please visit  https://www.pda.org/global-event-calendar/event-detail/rapid-microbiology-methods.

References

  1. USP<61> describes membrane filtration, plate count, and most probable number methods that can be done to quantitatively determine the bioburden of non-sterile DPs. Although 21 CFR 211.110(a)(6) does not specify a test method, it requires that bioburden in-process testing be conducted pursuant to written procedures during the manufacturing process of DPs.
  2. Sterility testing may be performed on the DS when it cannot be performed on the DP, as outlined in the final rule: Amendments to Sterility Test Requirements for Biological Products (May 3, 2012; 77 FR 26162 at 26165). Sterility tests are described in 21 CFR 610.12 and USP<71> Sterility Tests.
  3. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, July 1993.
  4. Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications, February 2010.

Friday, April 27, 2018

CRISPR Used for Infectious Disease Diagnostics

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) are short, repeated DNA sequences found in the genomes of bacteria and other microorganisms. These sequences help to fight off bacteriophages (bacterial viruses) by slicing the invading viruses, thereby preventing the virus from replicating.

CRISPR is now being used for a variety of applications. For example, CRISPR can facilitate the replacement of a mutant gene with the correct sequence, thereby curing a genetic disorder. CRISPR is now being explored to modify antibiotics that are specific for pathogenic bacteria without impacting good bacteria that the body requires.

The Harvard Graduate School of Arts and Sciences has developed an easy to understand website for reviewing CRISPR. It includes an illustration of the basic principles of CRISPR, shown below (click for a larger image):



Science Magazine has recently published an article on how CRISPR can be used for infectious disease diagnostics. Initial areas of focus target Zika, Dengue and human papillomavirus.

Different diagnostic methods are being developed which exploit the power of CRISPR technology. For example, double stranded (ds) DNA may be extracted from a sample and amplified using isothermal preamplification by recombinase polymerase amplification (RPA). The target sDNA amplicons are then sequence specifically cleaved by a Cas12a-crRNA protein complex, which activates nonspecific cleavage of single stranded (ss) DNA. A fluorescent dye binds to the ssDNA to create a detection signal. This process is known as DNA endonuclease-targeted CRISPR trans reporter (DETECTR).

HUDSON (heating unextracted diagnostic samples to obliterate nucleases) is a method by which heat and chemicals inactivate ribonucleases (RNases) and lyses viral particles, thereby releasing nucleic acids into solution. This can be combined with SHERLOCK (specific high-sensitivity enzymatic reporter unlocking), a Cas13-based nucleic acid detection method, to deliver a rapid (1-2 hour) diagnostic test. Using fluorescent readouts, the test can give a positive-negative readout on a paper test-strip. Read more about each of these methods, which are summarized in the illustration below (click for a larger image) with accompanying references, in Science Magazine.






Wednesday, February 7, 2018

Microbiology is Recognized with New Emojis

The Unicode Consortium today announced it has finalized a new set of 157 emoji. This brings the total number of approved emojis to 2,823.

The new Emoji 11.0 set is fixed and final, and includes the data needed for vendors to begin working on their emoji fonts and code ahead of the release of Unicode 11.0, scheduled for June 2018. The new emoji typically start showing up on mobile phones in August or September.

So, what does this have to do with microbiology or rapid methods? Believe it or not, there will be six (6) brand new laboratory and microbiology-related emojis including a lab coat, goggles, Petri dish, test tube, DNA molecule and finally, a microbe (which looks like E. coli on steroids!). These will be great additions to the microscope emoji which has been quite lonely for all these years!

Here is a sneak peek at the new emojis coming later this year. Please click on the image for a larger view.