Team:IISER Berhampur/Implementation

iGEM IISER_Berhampur
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Kit Design

Our kit will contain

  • Loop-mediated isothermal amplification (LAMP) reagents and phosphorothioate modified primers to amplify the DNA in the sample. Our primers will target TB-specific sequences of the rpoB and KatG genes. This will allow us to confirm the presence of genomic DNA of tuberculosis (TB).
  • In the second step, Cas14a1 nuclease, sgRNAs against Wild type or mutant RpoB and KatG genes, and a DNA probe containing Fluorescence Quencher (FQ) pairs will be used. The presence of a mutation will allow the specific sgRNAs to bind thereby triggering the nuclease activity of Cas14a and subsequent cleavage of the fluorescence quencher (FQ) pair. Cleavage will give fluorescence and the diagnosis will be positive.



Hardware Design

Non-Automated

To detect fluorescence with the highest efficiency. We are planning to use a low-powered laser, with a wavelength range between 480 nm to 500 nm, a mirror, an aperture, an interference filter, a sample chamber, and a mobile phone. The laser will act as a light source to illuminate the sample chamber so that fluorescence can be produced. Mirrors and the aperture will help us to adjust the intensity of light and help the light to fall uniformly on the sample chamber. The interference filter will filter out non-essential light (noise) to take up the maximum signal. The sample chamber will be a chip containing space where mutation detection and fluorescence production will happen. It will contain Cas14a1 along with LAMP amplified and T7 exonuclease generated ssDNA fragments and a DNA probe with an FQ pair that upon nuclease action would show fluorescence which indicates the presence of mutations corresponding to MDR-TB. The mobile phone will be an actual detector detecting the presence of fluorescence. The mobile phone will take multiple images and calculate the pixels present in the image and then average them out to give an output which is the presence or absence of fluorescence.

Automated

The automated design consists of three chambers.

  1. The pre-processing chamber
  2. The LAMP chamber
  3. Detection chamber

The device will be governed by software and the user will have to set up the time for each step manually.

  1. The user will have to insert a glass vial consisting of the sputum sample and glass beads into the pre-processing chamber. The vial will sit on a vortex that will run for a set time and complete the lysis.
  2. A suction tube will then suck the sample to another glass vial for isolation of DNA from the sample.
  3. The DNA will be introduced into the LAMP chamber for analysis. Details of LAMP chamber:
    1. The LAMP chamber is basically a vial inside a water bath already loaded with the LAMP primers and LAMP reagents, with a transparent screen on the top for observation.
    2. Once the sample is added to the LAMP Chamber, the water bath starts heating up to a temperature of 65-70℃, and the reaction continues for a set amount of time.
    3. After the reaction is completed, the device stops. The user will then have to observe for a colour change from the transparent screen.
    4. No color change indicates that the sample does not contain TB DNA and therefore the sample will be labelled as negative. The colour change indicates the presence of TB, next, the user has to confirm the presence of MDR-TB.
  4. For this, the user will turn on a switch, which will allow the machine to start detection of targeted mutations associated with MDR phenotype in Mtb. The suction tube will then suck the sample from the LAMP chamber and transfer it into the detection chip (detection chamber). The detection chip will be loaded with T7 exonuclease, cas14 (in power form) + guide RNA complex and FQ pairs.
  5. After a set amount of time, the laser will be switched on, and the beam will be directed to the chip. The detection of fluorescence will prove the presence of MDR-TB in the given sample.
  6. The components in the chip, the pellet tube and the sample collection tube are then discarded before the next detection cycle.



Our proposed end users

In low income countries like India, detection for MDR is unavailable in peripheral regions like in rural areas and is only available in the far places from the towns. Financially challenged patients usually cannot afford doctor’s advice of proper diagnosis and consequently can potentially spread MDR-TB.

  • Our proposed kit will be cost-effective and capable of fast detections which can be easily affordable by clinics and diagnostic centres in rural areas and small towns.
  • Our designed kit can easily be implemented in labs with less facilities.
  • The kit also provides a point of care diagnosis where patients who cannot afford a doctor’s appointment can get their samples tested at Primary Health Clinics.
  • We plan on commercializing our system in the form of an automated hardware device which will enable us to maximize our reach to such clinics and diagnostic centers simultaneously allowing bigger hospitals to utilize this as a cheaper means of diagnosis.



How will diagnosis centers and clinics use our

project?

User Guide:


Sputum sample collection from patients

Sputum should be collected in small cups provided to the patient.The patient should be advised to breathe in a few times, cough hard, and spit out the sputum in the cup.

Extracting bacterial DNA from the sputum sample


DNA isolation protocols for bacterial DNA isolation must be used to lyse the cells and isolate bacterial DNA. Mechanical methods like sonicator/beads can be used to lyse cells followed by using reagents that can precipitate DNA.

LAMP assay

Loop-mediated Isothermal Amplification should be performed on the extracted bacterial DNA following the provided protocol. One set of phosphorothioate primers designed for wild type and mutant (provided in the kit) tuberculosis should be used for the subsequent generation of single-stranded DNA (ssDNA) (see below). This assay will confirm the presence of TB DNA if color change occurs. If the assay shows no color change results can be given as negative.

Generation of ssDNA from LAMP amplified DNA

T7 exonuclease will cleave DNA from 5’ to 3’ end but will be inhibited if the 5’ end has a phosphorothioate modification. By using one set of phosphorothioate primers in the LAMP assay, we generate one strand in each amplified DNA with phosphorothioate modification, which will be resistant to T7 exonuclease digestion. The other strand will be digested by T7 exonuclease.

Reaction of Cas14a1 with ssDNA, sgRNAs and DNA FQ probe

•To the LAMP positive wells, treatment with T7 exonuclease will be followed by addition of cas14a1, sgRNAs. Separate wells should be used for the single guide RNAs specially designed for mutated strains of KatG and RpoB.
 •To this solution, fluorescence quencher pairs should be added. The presence of mutations corresponding to MDR-TB will allow sgRNAs to bind which will activate the trans cleavage activity of cas14a1 to cleave the FQ pair generating fluorescence.
Be careful to sterilize your surroundings to prevent contamination. Make sure that the equipments being used are autoclaved before use. (This step is being done to confirm the presence of MDR-TB)

Detection

A mobile phone camera should be placed on top with the camera facing the opening. The software device on the phone will detect the fluorescence and generate the report. Fluorescence is detectable by the naked eye but for standardization, we recommend using a mobile phone camera to record the readings.

Result Analysis

If the fluorescence detection comes negative but LAMP is positive, it can be inferred that the TB is wild type and not MDR-TB.

Automation

For the automated device, the user will only have to collect the sputum sample and pour this into a tube which will go into the first compartment of the automated device.





How would you implement your project in the real

world?

Implementation is essential for any good research. It allows us to see the plans become a reality. We envision assembling an automated diagnostic tool kit that will not only detect TB but also MDR-TB. Our proposed design is portable, cost-effective, and doesn't require skilled workers. By commercializing our innovation coupled with marketing and advertising, we aim to maximize our reach. Our device will be accessible to people in urban areas with big hospitals as well as in rural areas with poor infrastructure and small diagnostic centres. The idea of eradicating TB lays the foundation of our project. Achieving it requires us to spread awareness among the general public regarding this dreadful disease. Educating people about the requisite precautions and the necessity for professional help is the need of the hour. Doing this will not only make them more aware and cautious but also increase the number of cases that are detected and given the right treatment. This will, in turn, reduce the mortality rate as well as the spread of the disease, thus saving thousands of lives. Increased detection would require the need of handy and affordable diagnostic tools like ours. Not only this, it will help the doctors to give the proper treatment at the right time as our kit will also detect MDR strains. All this will finally allow us to achieve our ultimate goal of "TB Eradication". Our proposed model has a highly impactful real-life application.



What are the safety aspects you would need to

consider?

Medical diagnostics devices are regulated by CDSCO. The regulations are tight and require analysing biosafety and biosecurity aspects. Biosafety includes ensuring that the synthetic biology of our project doesn’t harm any individual or environment. Biosecurity aspects usually deal with keeping the biological product safe from misuse.
Throughout the design process we identified the risks associated with our project and tried to address them.
To know more about Safety please visit - safety page.



What other challenges would you need to consider?

We are still at the initial stages of developing and implementing our proposal. Definitely, there are a few challenges we have to consider seriously while implementing our proposal. With several interactive sessions, interviews with many stakeholders and patients, we came to know about the biggest hurdle in the path of eradicating MDR-TB. There is a lack of information and knowledge about MDR-TB among people. It's necessary to educate people first about MDR TB, its symptoms, and treatment, then we have to maximize our reach to as many patients as possible. We have to reach out among common people and show them the benefits of using our test kit over conventional practices!

One of the unique selling points of our proposal is the cost-effectiveness of our kit. People will prefer our kit if it is accurate as well as cheap in comparison to methods in use today. Our next challenge is to make sure that the cost of our kit does not increase while we go through different stages of manufacturing.

Biosafety is another big challenge that we have to encounter very carefully. There are several instances where there is a lack of safety while handling the test samples or while performing tests. This could lead to a severe outbreak of MDR-TB. We have to make sure that the samples are handled and the tests are performed in an utmost safe and secure environment with proper PPE and adhering strictly to protocols. We will be making a manual that will have all the protocols and safety regulations that have to be followed while using the kit.