Team:CCU Taiwan/Description
Inspiration
Outbreak of an epidemic disease may ruin a country, both economically and psychologically, even if the disease affects animals but not people. For example, Foot-and-Mouth Disease (FMD, Aphthous fever) was detected in Taiwan on 19 March 1997, resulting in culling of 3,850,536 pigs by July of the same year. This incident not only broke the hearts of pig farmers and agricultural workers but also become a nightmare for other Taiwanese, who witnessed on news reports the burial and incineration of thousands of pig carcasses.
During the 20th century, many diseases have been spreading widely, causing devastation around the globe. During our brainstorming session in Dec 2018, numerous pig carcasses floated onto the coastline of Kinmen, Taiwan, and tested positive for African Swine Fever Virus (ASFV). This event serves as a reminder of the severity of the ASFV plague in the Asia continent. Since then, the plague has spread across East and Southeast Asia.
In Taiwan, pork is an important part of the daily diet. Currently there are around 5.5 million pigs in Taiwan. If the plague were to spread to Taiwan, the effects on Taiwanese society and the domestic economy will be unmeasurable, considering the massive dependency of the population on pork products. Not only animal husbandry would be affected, but this would cause a chain reaction in animal fodder, transportation, food processing and even the meat export industries of the country.
To prevent this tragedy, we decided to develop a device to prevent a widespread epidemic by early detection of ASFV. Thus, we have designed an automated, portable, user-friendly robotic testing instrument called ASFAST. ASFAST is capable of detecting ASFV on the spot and uploading the results immediately to an online database, to allow rapid containment of any outbreak. The machine provides a minilab employing the specialized functions of the CRISPR-Cas system and PicoGreen. The CRISPR-Cas system will allow viral DNA detection, while PicoGreen is an intercalating fluorochrome to amplify signal expression.
Moreover, we hope to raise world awareness of the problem through this initiative. By showing bravery on the journey of overcoming obstacles while conducting the project, even though we are nobody, we hope to inspire others to do even more.
During the 20th century, many diseases have been spreading widely, causing devastation around the globe. During our brainstorming session in Dec 2018, numerous pig carcasses floated onto the coastline of Kinmen, Taiwan, and tested positive for African Swine Fever Virus (ASFV). This event serves as a reminder of the severity of the ASFV plague in the Asia continent. Since then, the plague has spread across East and Southeast Asia.
In Taiwan, pork is an important part of the daily diet. Currently there are around 5.5 million pigs in Taiwan. If the plague were to spread to Taiwan, the effects on Taiwanese society and the domestic economy will be unmeasurable, considering the massive dependency of the population on pork products. Not only animal husbandry would be affected, but this would cause a chain reaction in animal fodder, transportation, food processing and even the meat export industries of the country.
To prevent this tragedy, we decided to develop a device to prevent a widespread epidemic by early detection of ASFV. Thus, we have designed an automated, portable, user-friendly robotic testing instrument called ASFAST. ASFAST is capable of detecting ASFV on the spot and uploading the results immediately to an online database, to allow rapid containment of any outbreak. The machine provides a minilab employing the specialized functions of the CRISPR-Cas system and PicoGreen. The CRISPR-Cas system will allow viral DNA detection, while PicoGreen is an intercalating fluorochrome to amplify signal expression.
Moreover, we hope to raise world awareness of the problem through this initiative. By showing bravery on the journey of overcoming obstacles while conducting the project, even though we are nobody, we hope to inspire others to do even more.
African Swine Fever Virus
In 1907, a fatal infectious disease struck swine in Kenya, Africa. The virus which causes this fatal disease was identified in 1921 and given the name African Swine Fever Virus (ASFV), based on the location of discovery and the host. When ASFV infects swine, the virus will incubate inside the body of swine for 4-19 days. After incubation, the virus starts to attack leukocytes and paralyses the immune system, resulting in acute hemorrhagic disease. The mortality rate of the infected hosts is a staggering 100%. Unfortunately, authorities are able to take action only after they are notified by the pig farmers of a mass death of pigs. Due to the late response to the incident, the virus might have spread widely, leading to inefficient control of the epidemic.
The spread of ASFV
In addition to the delayed response of swine to ASFV infection, the ASFV itself is highly infectious, partially based on the fact that ASFV is extremely stable and infects a new host through multiple ways. ASFV is highly resistant to low temperature, but may be inactivated by 56°C/70 minutes or 60°C/20 minutes. The virus can survive from pH 4 to pH 13. The paths of ASFV infection include all forms of discharge by the host, equipment and other objects including food that have come into contact with an infected host. Since 2005, a total of 50 countries have reported encountering ASFV, ranging from countries neighboring Kenya to some as far away as China. The spread of ASFV is mainly caused by the transportation of infected swine and its secretions, and also pork products.
Detection of ASFV
Currently, the common method for ASFV detection requires that a blood sample be sent to a lab to undergo an ELISA test or PCR. An ELISA test detects whether the blood contains antibodies to the ASF Virus, which means that infection can only be detected by this test after the incubation period. On the other hand, PCR is a time-consuming process which requires DNA of ASFV to be copied until there is enough of it for analysis and detection, usually by running on an agarose gel and comparison with a positive control. Importantly, the transport of blood samples may cause unnecessary risk of virus spread. Thus, we came up with a new idea of developing a rapid screening device, to allow epidemic prevention authorities to do rapid medical tests before the pigs show symptoms.
CRISPR-Cas12a
CRISPR technology has provided a quantum-leap in this new era of molecular biology as it is capable of removing, adding or altering genes within organisms. CRISPR-Cas system is a prokaryotic defense mechanism against foreign genetic elements, including double strand (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). Before target recognition, the Cas protein will bind with crRNA (CRISPR RNA), which contains the T-rich protospacer adjacent motif (PAM) sequence, to form a Cas-crRNA complex. Once the target recognition is completed under the guidance of the PAM sequence by Cas-crRNA complex, target editing or cleavage may occur. In our project design, we use CRISPR-Cas12a in our screening device to detect an ASFV sequence. CRISPR-Cas12a is a type V CRISPR protein that cleaves target DNA. The most significant aspect is that it also possesses discriminate single-stranded DNA threading activity. This specificity accesses our signal expression and amplifying protocol after detection of its target genome by using PicoGreen.
African Swine Fever Autonomous Screening Technology (ASFAST)
To detect ASFV in an early phase of infection with high correctness and low risk, we decided to develop ASFAST (African Swine Fever Autonomous Screening Technology). ASFAST is based on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology and PicoGreen fluorescent dyes. We plan to use a specialized member of CRISPR family, Cas12a, as the key component to detect the dsDNA sequences of ASFV genome.
ASFAST will detect ASFV through a four steps process:
ASFAST will detect ASFV through a four steps process:
- In the first step, Cas12a protein will assemble with ASFV-specific crRNA to serve as an ASFV-specific detector. Once the Cas12a-crRNA complex recognizes the ASFV dsDNA sequences in samples, the trans-activity of Cas12a of ssDNA digestion will be activated.
- In the second step, the trans-activated Cas12a-crRNA complex will digest the ssDNA pre-conjugated on magnetic beads in the same solution.
- In the third step, the naked magnetic beads will be transfected to a new tube by an electromagnet. The PicoGreen fluorochrome and ssDNA complementary to the ssDNA pre-conjugated on magnetic beads will be added to solution.
- In the last step, the PicoGreen fluorescence signal will be measured by fluorometer, and the collected data will be analyzed and transferred to a server via wireless network.
- Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018).