According to data from a study published by NEIN in 2017, more than half of the world's population is infected with H.pylori. And according to the Fifth National Consensus Report on the Management of H.pylori Infection, the current prevalence in Asia is 54.7%. H.pylori infection will further trigger a variety of gastric diseases, including gastric cancer. The mortality rate of gastric cancer is usually related to the stage of gastric cancer. Patients with early and middle-stage gastric cancer can be detected and diagnosed, they usually can obtain a certain cure rate and a relatively low mortality rate. (Xiaomei Zhang, 2022)
Our team launched a survey called 'Stomach! How are you?', which has a sample size of 408 and is distributed to various aged respondents. 34% of our participants admitted they were not familiar with the detriment of H.pylori, and more than half of the respondents claimed they haven't done any H.pylori test.
Early screening and early treatment for H.pylori could reduce gastric cancer incidence and mortality. The "Expert Consensus Opinion on Early Stomach Cancer Screening Process in China (Draft)" issued by the Chinese Anti-Cancer Association in 2017 recommends regular gastric cancer screening for people over 40 years old. We realized that there still exists a great market for H.pylori detection.
Therefore, we hope to develop a kit that can be tested quickly and easily extended to more people and contribute to human health.
Compared to the current H.pylori assay kit and the C13/C14-breathing test in the market, our product features high accuracy, low-priced, portable, and no special instruments are required. Our product can compensate for the needs in the current market for effective self-testing kits and make H.pylori detection more convenient without bothering to go to hospitals every time.
Per our previous investigation, actually, everyone shall be our potential end users, especially those who have bad diet habits easily exposed to H.pylori infection, and those family members of the H.pylori infected.
In order to make our end users access to our products, we have targeted some stakeholders with who we could cooperate to promote the use of our H.pylori infection assay kits to broader end users. Following we categorize them into three types:
Compared to the carbon-breath test that public hospitals offer, our product is more convenient to use at home by individuals. Since state guidelines recommend that all adults over 18 are tested and eradicated for H.pylori, and both a trip to the hospital and the carbon-breath test are inconvenient, cost-driven, and time-consuming, our product could be used in advance for follow-up treatment to free patients from the inconvenience of traveling back-and-forth.
Considering the fact that poultry may transmit H.pylori from livestock to humans, putting breeders and customers at risk. In animal husbandry or on livestock farms, livestock such as omophagia and poultry have a high prospect of being infected with H pylori with infection rates much higher than the average human infection rate of 59%. Animal husbandry also needs a massive amount of testing kits, hence our product with its features at a low price, massive production and self-testing will take advantage of this market.
For restaurant chains, the pain point lies within the need to guarantee their hygiene in the process of storing, cooking, and serving. It's hazardous for consumers to eat contaminated food, discrediting restaurants' reputations too. Moreover, the catering industry needs a massive amount of testing kits if they want to confirm whether every staff even food is infected by H.pylori or not, but the cost is often hard to afford. With our low-priced product, restaurants can regularly examine their foods and staff for H.pylori infection to ensure the food hygiene for their customers.
Figure 1. Prototype of our H.pylori infection assay kit and its operation procedure
Note：
-Do not eat, smoke, chew gum or drink for 30 minutes before taking a saliva sample.
-Prochip series is embeded with microchip on the basis of the classic series
Our team aims to develop an in vitro reaction system for H.pylori detection with FnCas12a. In the laboratory, we synthesized four DNA fragments of H.pylori characteristics genes as targets and used the purified FnCas12a protein to recognize and cut those target genes. Finally, we designed a reporter system to easy detection the activity of FnCas12a and then measured the fluorescence intensity to confirm the detection result. The below figures illustrate the principle of our in vitro H.pylori detection platform and the steps to complete our experimental design.
Figure 2. The principle of our in vitro H.pylori detection platform and our experimental design
Figure 3. Fluorescence intensity at different concentrations of oligo DNA in simulated bacteria.
NC = negative control
As shown in figure3, as the concentration of oligo DNA increased from 0 ng/L to 4 ng/L, the fluorescence intensity of the ssDNA fluorescent probes of the ipaH, invA, cagA, and 16S systems also increased significantly. Thus, the results indicate that our fluorescent detection experiment is successful. Cas12a protein and sgRNA can recognize and cut the oligo DNA probes and the fluorescence emission can be detected.
In our experimental work, we only use one type of bacteria in the experiment: E. coli BL21(DE3), a non-pathogenic strain of microorganisms. This is due to the biohazard applications H.pylori, S. Typhimurium, and S. flexneri pose. However, in consideration of the environmental and ecological issues, all substances contaminated by E. coli BL21(DE3) will be sterilized before disposal and will be discarded in the biohazardous bin. Our experiment doesn’t consist of any human or animal parts or genes. And all genetic modifications that were established in our process are limited to research purposes.
Regarding our final product, since it is an in vitro detective tool, will not exhibit addictiveness or exclusiveness. The major concern is the manipulation of H.pylori which belongs to the third group of pathogenetic microorganisms. Therefore, it will be important to ensure that the H.pylori samples are all sealed and transported, strictly following “Regulations on biosafety Management of pathogenic Microbiology laboratories” published by Order of The State Council of the People's Republic of China No. 424.
Regarding the commercialization of our products, if we want to implement our product in hospitals and clinics, we need to consider how our method could supplement current methods so as to highlight its selling points. Since hospitals and clinics exhibit reliance on current methods, lacking in incentive to look for better alternatives. This might result in a reluctance to accept our product. Quoting Dr. Li from our expert interview, hospitals “already have sufficient methods for H.pylori detection”.Listed below are the potential solutions we have proposed to overcome this challenge:
1. Comparison.
We have not yet compared the sensitivity and specificity of our test with those of currently available
methods. We will conduct experiments to compare the performance of our CRISPR Cas-12a detection with other
clinical methods to find our strength, probably in accuracy, rapidity, and sensitivity.
2. Differentiating H.pylori strains.
The toxicity of H.pylori varies significantly between different strains. Thus, it’s important not only to
detect the presence of H.pylori but also to differentiate the strain. Currently, separate differentiation
tests must be performed outside of H.pylori detection, which is time-consuming and costly. Thus, current
hospitals and clinics do not provide the service to differentiate the strain in infected patients.In contrast,
our method is already able to differentiate between two different sequences in H.pylori, which means our
method can differentiate H.pylori strains rapidly and concurrently complete basic H.pylori detection. In the
future, we will design different sgRNAs for our CRISPR Cas-12a system to differentiate more types of H.pylori
strains to make our products more outstanding.
3. Convenience as home kit.
To implement our product in each home which means no special instrument is required, we need to develop a new
method to replace PCR and visualize the fluorescent result. Per the suggestion we obtained from the expert
interview, we applied the isothermal amplification method to achieve the function of gene amplification which
only requires hot water. Regarding the visualizing results, we will further study lateral flow strips for
displaying the results positive or negative. Quoting Ms. Su from our street interview, "if your product can't
be as convenient as the COVID-19 antigen test, I won't consider buying it". Thus, it is imperative that we
must find more convenient substitutes for our current experiment procedure.
To overcome the challenges per the previous speaking, we plan to optimize our experiment through the following method:
1. Use LAMP as the substitute for PCR. LAMP allows for isothermal amplification of target sequence without PCR instrument, only 65 degrees' hot water is required for 30-60 minutes. But it still needs us to conduct another round of surveys to investigate the willingness of the potential users to operate this.
2. Use LAMP as the substitute for PCR. LAMP allows for isothermal amplification of target sequence without PCR instrument, only 65 degrees' hot water is required for 30-60 minutes. But it still needs us to conduct another round of surveys to investigate the willingness of the potential users to operate this.
In the meantime, we also plan to introduce the following features in our products in response to yet unanswered demand in the market:
We have drafted a business plan for implementing our business in more detail, including marketing strategy and promotion plans which you can find on the Supporting Entrepreneurship page.
