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Implementation

Overview​

To build a universal disease diagnosis platform based on polymerase mediated technology


In China, only 26% of primary care doctors correctly diagnose patients according to a 2020 Lancet study. At the same time, antibiotic overuse is common, far exceeding WHO recommendations. This misdiagnosis and over-prescription of antibiotics is a global health challenge, causing at least 700,000 deaths per year according to a 2019 WHO report.
- Therefore, we aim to develop a new diagnostic method to improve diagnosis and curb antibiotic overuse.

Our team around the polymerase mediated chain displacement reaction design developed weighted and sum calculation of DNA molecular probe, artificial biochemical circuit of neural network structure, can perform weighted sum multidimensional biomarker input signal, thus output more intuitive low-dimensional assessment results, used for in vitro diagnosis and identification of disease types.

  • In this technology, the innovative design of molecular probes with polymerase, which not only reduces the error caused by leakage, improves the accuracy of the system, but also redesigns the weighting process to reduce the design and manufacturing cost of the weighting chain, greatly reduces the complexity of the reaction, and makes molecular diagnosis of high-weight targets possible.

  • In the future, this technology is expected to build a universal disease-accurate diagnostic platform, achieve more accurate new diagnostic methods, help doctors accurately diagnose and treat diseases, and benefit patients around the world.

Proposed end users​

As an auxiliary diagnostic tool, the polymerase technology-based precision testing platform can be used for doctors' initial judgement on the one hand, and then accurately identify whether a disease is present or not through the technology on the other hand.

  • This method is based on the perspective of DNA/RNA micro-level changes to detect and diagnose, and the macro-expression of confirmation, thus optimising the diagnosis time of existing diagnostic techniques.

  • Additionally, the polymerase-based precision testing platform can also monitor disease recurrence in patients who have recovered from their disease. For some recovered cancer patients, our precision testing platform can predict the risk of cancer recurrence at an early stage through the microscopic level of DNA/RNA.

Therefore, the recommended users of our diagnostic tests are two-fold:

  1. Patients referred by their physicians for suspected disease, who can use our platform for initial diagnosis guidance.
  2. Patients who have recovered from disease and need monitoring for recurrence, where our platform can predict relapse risks early through microscopic DNA/RNA analysis.

Envision​

As pioneers of precision diagnostics, our vision is to be able to screen for the exact type of disease at an early stage in order to plan further treatment.

  • We promote the use of precision testing platforms for certain targeted blood tests, and it is then up to the physician to correlate the macro symptoms with the micro results to determine whether the patient should be referred for further invasive testing.

Implement​

Design detection probes​

  • First, after selecting the disease to be detected, the appropriate target genes in the database and their importance in the detection process (weight) are selected.
  • Then, the detection probe for each target is designed and the function of the probe is characterised to see if it can be weighted.
  • Finally, we simulate real samples and experimentally prove that the molecular classifier composed of our probes is capable of realizing calculation and classification at the molecular level.

Actual diagnostic process​

First, serum samples from a sufficient number of patients were extracted, RNA in the serum was extracted, and the cDNA was reverse transcribed.

Then by LATE-PCR amplification concentration, adding the probes for molecular calculation, the target DNA and one end of the weight probe by base complementary pairing, followed by polymerase catalyses the subsequent DNA replication strand displacement reaction.

  • The free fluorescent chain releases the fluorescent signal to realize the qualitative detection of the target sequence, thus showing the bands of different colours and different depths in the test kit after dropping into the patient's blood sample, which is the initial completion of the process of detecting the presence of cancer factors in the test sample.

At the same time, different numbers of weight chains are released during DNA polymerisation, so fluorescence signals of different intensities are released during this step reaction, i.e. they correlate with the amount of target miRNA present in the patient's blood sample, and the amount of target miRNA in the sample will eventually reveal the cancer probability of the patient being tested.

  • Therefore, by detecting the fluorescence signal, the data of the measured fluorescence signal is analysed, processed, and the visualisation software is used to make the results clearer and more readable, and the diagnostic results are obtained.
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Figure 1, A Schematic Diagram of the Diagnostic Process of the Universal Disease Precision Diagnostic Platform Developed Based on Polymerase Mediated Technology

The diagnostic process of our disease precision diagnosis platform will not endanger patients' health. The test only requires a few blood samples, which are easy to collect, and the molecular computing system of the project is simplified.

  • The construction of the probe is more economical and simple, and the downstream computing module is easily accessible. The examination of patients is more convenient and accurate, which reduces the cost of medical treatment and the economic pressure on patients.

Safety considerations​

If whole-cell biosensors belonging to genetically modified organisms are used in this project, the potential societal risks and associated biosafety issues will mainly be ecological safety issues, biological defence issues and conceptual ethical issues.

  • GMOs and Biosafety From a safety point of view, the entire process from input to output of our project is carried out in a cell-free system belonging to a cell-free sensor and does not involve living individuals.
  • Patient Safety Our reagent kit does not put patients at risk and only blood samples need to be taken. It should be noted that the test relies on certain professional instruments and therefore needs to be performed in appropriate facilities, such as hospitals and dedicated testing facilities, and by trained personnel to achieve more accurate testing.
  • Cross contamination During the production and testing processes, products are prone to cross-contamination, which may affect the detection accuracy. Therefore, the production of products must strictly control the environment and personnel hygiene and safety conditions, and the product quality must be strictly controlled by professional personnel.

To solve the security problem, We have therefore contacted experienced individuals such as Dr Yi Cai from the General Hospital of the People's Liberation Army of China. Please refer to the 🔗Practice page for relevant discussions.

Challenges​

We are still in the early stages of developing and implementing our proposal. However, there are a number of challenges that we need to seriously consider in order to make our system work better.

Molecular diagnostic kit challenges​

Since the accuracy of the molecular diagnostic test kit is affected by many factors such as sample quality, operator skill, kit quality and so on, false negative and false positive results may occur. This may lead to the disease being missed or misdiagnosed, which may affect the effectiveness of treatment.

  • In addition, the instrument has high precision, high technical requirements for experimental operation, and may require professional laboratory equipment and technical personnel to operate, so the detection cost is relatively high.

  • Most importantly, there may be biomarker concentrations in serum samples that are lower than the sensitivity of the assay and may be misinterpreted as negative, leading to inaccurate predictions and no effective assessment of disease progression and prognosis.

Sample data challenges​

As an early screening diagnostic tool, we would like to learn more about potential marker relationships in serum samples.

However, we are currently using a database of tissue samples, which may lead to less specific detection results due to the influence of tissue source and sampling site on the molecular markers in the tissue samples.

Technical and system challenges​

Due to the complexity of the reaction system, more precise control and optimisation of reaction conditions, buffers and enzymes are required to achieve efficient nucleic acid amplification and accurate detection results.

There is also the challenge of standardisation and quality control of reaction systems to ensure that results are comparable and reproducible across different experiments.

Our next step is to combine the advantages of high throughput, high sensitivity, high precision and high automation of microfluidic chip technology to eliminate the nucleic acid amplification step and improve accuracy.