Overview
In China, echinococcosis mainly prevail in the pastoral areas and semi-pastoral areas of northwest provinces.
At present, echinococcosis is mainly diagnosed by imaging or immunological methods,which can only be used in the middle and late stages. Moreover, these methods are in high demand for instruments and technical personnel, which are not easily available in many epidemic areas. Therefore, a simpler and faster method is needed for early detection of echinococcosis.
Our project is aimed at making a miniaturized kit based on RPA and CRISPR-Cas12a. It is designed detect the cfDNA of echinococcus for the diagnosis of early echinococcosis. We believe that, compared with traditional detecting methods, a simpler and more convenient diagnostic kit is more acceptable in regions with limited medical resources.
Summary
As a parasitic disease that is difficult to detect in vitro, hydatid disease has always been a living problem for many herdsmen in Tibet. In order to solve this problem, we have designed this "sniper" to clear them out of the problem.
Fig 2. Product concept diagram
SNIPER E is designed as a class of kit that can quickly detect the presence or absence of cfDNA in the blood. We want our kit to be complete as follows:
1.Blood can be taken and cfDNA be quickly extracted
2.The cfDNA was rapidly amplified with RPA
3.The amplified products were specifically digested by the Cas12a protein
4.Show the results on a test stripe
The implementation of instrument-dependent kits cannot achieved without the completion of each small step. How to implement this kit? How can we ensure its safety? We will explain these issues in detail below.
Operational pattern
Compared with commonly used PCR technology, the RPA technology used in the kit has lower requirements for testing equipment. This kit can be used at room temperature, and the needed detection time is shorter. More importantly, the amplification steps using RPA are very simple compared with traditional PCR, even untrained personnel can get started with simple training.
Table 1. The superiority of RPA
Similarly, the CRISPR system used in the kit has good specificity and sensitivity. Compared with traditional detection methods, it does not require many complex instruments, such as high-throughput sequencers and qPCR machines, which also enhances the usability of the kit.
Fig 3. Test strip detection principle
The kit we designed is not an instrument-dependent kit, and we can complete the detection of hydatid cfDNA in a thermos cup, and show the results as a test strip.
How can we achieve this goal
Blood was collected from patients and the cfDNA was quickly extracted by using the kit reaction unit
The cfDNA was placed into the RPA reaction unit to amplify
The amplified products were placed into the detection reaction unit
The test strip was inserted into the test reaction unit to wait for 10 minutes to observe the results
Protocol
In consideration of storage and transportation, the kit is wrapped with an ice pack. The kit is made of thermal insulation material, which can keep the temperature constant during the reaction.
Our kit is divided into five parts:
1. Needles for blood collection, infusion tubes, and tubes for storing blood.
2. There are three tubes for rpa, including a dry powder tube, which contains three proteins just like the tubes used in our experiment, but the mixed primers are also made into dry powder and added. The second tube contains Buffer A. The third tube is filled with Buffer B and diluted with DEPC water (because the amount of Buffer B is small, it is inconvenient to fill it alone).
3.Cas digestion reaction tube, containing all components except rpa product, directly made into liquid tube (skip pre-incubation)
4. Test strip part
5. Disposable pipette for liquid transfer
Method of use
1. When used, the blood collection equipment should be used to collect the blood of the person to be tested
2. Use the pipette according to the instructions to take Buffer A and add it into the dry powder tube, then add the sample blood, mix and then add Buffer B and mix again
3. The reaction tube was immersed in 39℃ water for 30 min
4. Take the product after the reaction, add it to the cas enzyme digestion reaction tube, and mix it well
5. The reaction tube was immersed in 37℃ water for 30 min
6. Take the test strip and infiltrate it into the enzyme digestion reaction tube. The results will be displayed after 5 min, and the detection results will be credible within 15 min.
Target Users
Compared with traditional diagnostic methods, the main goal of diagnostic reagents and design is to be convenient, fast and easy to use. The purpose of this design is to provide hydatidosis-endemic areas with the means to carry out early detection under limited medical conditions.
In small community hospitals, health centers, mobile epidemic prevention stations, etc., detecting kits can help doctors quickly diagnose hydatidosis, thus providing more timely treatment for patients.
At the same time, the kit is also a good supplement and proof of traditional diagnostic methods. It can be used in combination with other diagnostic methods for higher diagnostic accuracy and efficiency.
For residents of remote pastoral and semi-agricultural and semi-pastoral areas, detecting kits are also a good self-testing method that can quickly help patients detect hydatidosis and seek further medical assistance depending on the specific diagnostic results.
Fig 4. Infection with echinococcosis in China
Security
The test method we designed is non-invasive, only the blood collection will contact with the patient, and no other steps will not contact with the patient, ensuring the safety of the kit.
Cost
The research and experiments that we have done or will do in the laboratory are not cheap, but after we complete the research and development of all the technologies, the cost of manufacturing this kit will not be very high, which can meet the needs of herders.
