Team:Shanghai HS United/Description
African swine fever (ASF) is a highly contagious and deadly viral disease of pig that has casted a serious threat towards pork industry worldwide. In 1921, ASF was first discovered in Kenya, Eastern Africa. In the 1950s, it rapidly spread across Europe including Spain, Portugal, Italy and France. But in the mid-1990s, ASF was eradicated from Europe, with the exception of Sardinia. Unfortunately, in 2007, ASF once again broke out, in large scale, globally, successively appearing in eastern Europe, Russia and other parts of eastern Asia. In March 2017, an outbreak of African swine fever occurred in Irkutsk state in Russia's far east, which is close to China. China's first case of African swine fever was diagnosed on August 3, 2018. Until July 11, 2019, ASF has appeared in all major provinces and cities in China, causing huge and irreversible economic losses in the whole pig product market.
A country that eats more pork than any other on earth and where even lower cost chicken trails well behind pork in popularity (though yes, it is sneaking up a bit like everywhere in the world) is an export target for sure. It is understandable how it might consume the most total pork compared to other countries since it has the largest population. However, that's not the real story. The heroic part is notwithstanding the wide range of incomes still present in China, and the dramatic middle class expansion of the last two decades, Chinese pork consumption is estimated to be over 30 kg per person per year, roughly equal to the European average consumption and over 30 percent more than US per capita consumption. They achieve this feat with incomes roughly 20% of the average EU income and 14% of average US per capita income. What could go wrong here for the exporters? See Figure 1. Note that the rate of growth of pork consumption has been falling for several years in China and is correlated to the same fall in the rate of real growth in Gross National Product (National Income) GDP.
For ASFv early diagnosis, it should only be detected in specialized laboratories with current techniques. Our team modified the Cas12a detection method, which combined with LAMP amplification and lateral flow. Our experiment has discover the most effective set of conditions that would allow the entire detection system to be rapid, simple, and accurate through the use of test papers. However, there are certain disadvantages in our method: even with the addition of UDG, the final detected results still show evidence of contamination. To prevent aerosol contamination, we specially designed our kit with a simple device to be as enclosed as possible to avoid exposure to the atmosphere.
- bluish-purple areas and haemorrhages (spot-like or extended) on the ears, abdomen, and/or hind legs;
- ocular and nasal discharge;
- reddening of the skin of the chest, abdomen, perineum, tail, and legs;
- constipation or diarrhoea, which may progress from mucoid to bloody (melena);
- vomiting;
- abortion of pregnant sows at all stages of pregnancy;
- bloody froth from the nose/mouth and a discharge from the eyes;
- the area around the tail may be soiled with bloody faeces
Due to the very short time of infected pigs from morbidity to death, a large proportion of them are diagnosed long after the death. Therefore, the rate of potential disease transmission increases dramatically.
ASFV can be mainly transmitted in the following ways:
Firstly, it can be transmitted through direct contact with blood, feces, saliva, urine of infected pigs or contaminated environment, tools and food.
Secondly, transport of pigs, transport infected pigs is also a main way for the rapid spread of the virus. For example, in 1964, the debut of ASF in France was caused by the illegal transfer of pigs, some of them were infected by ASFV, from Spain.
Thirdly, contact of contaminated substances. It can be found from the history of ASF's diffusion that pig products and slops are also the main factors for the spreading of the virus across regions. For instance, the ASF, introduced to Portugal in 1957, was proved that it was caused by pigs which near Lisbon ate the shipping waste from Angola.
Fourthly, wild boars and soft ticks. Both wild boars and soft ticks can be infected by ASFV, and the virus can be transmitted from tick to tick by laying eggs or mating. Ticks, although, have a small range, less than a few tens of meters, the virus can be spread over a long distance with the movements and migrations of hosts, such as wild boars. For example, the occasion of ASF in South Africa in 1928 was speculated to be related to the contact with domestic pigs and warthogs as well as ticks.
Since each animal could be at a different stage of the disease, both virus and antibody detection tests should be carried. To ensure the accuracy, the testing process for African swine fever (ASF) can only be taken in nationally certified laboratories. The followings are some internationally recognized testing methods:
ASFV genome detection by polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is used to detect the ASFV genome in porcine samples (blood, organs, etc.) and ticks. Small fragments of viral DNA are amplified by PCR to detectable quantities. All validated PCR tests allow viral DNA detection even before the appearance of clinical signs. Although PCR provides a sensitive, specific, and rapid alternative to virus isolation for the detection of ASFV, it required hours to diagnose ASF as the sample arrival to the laboratory.
ASF antigen detection by antigen ELISA test
Viral antigens can also be detected using ELISA, which is cheaper to set up than PCR methods and allows large-scale testing of samples in a short time without special laboratory equipment. However, as in the case of the FAT, in subacute and chronic disease the antigen ELISA has a significantly decreased sensitivity. In addition, field samples are often in poor condition and therefore also decrease the sensitivity of the test.
In conclusion, current available diagnostic tests allow one to confidently diagnose ASF by combining both virus and antibody detection. Real-time PCR is the most widely used for virological diagnosis, providing sensitive, specific, and swift detection of ASFV. Every of them, nevertheless, has more or less drawbacks during the testing process. Therefore, we aim to find a perfect testing method can meet the high accuracy, high sensitivity, high specificity and high efficiency at the same time.
In order to attain a highly effective and efficient detection method, we finally choose to use Loop-mediated isothermal amplification(LAMP), Cas12a Protein and Lateral Flow dipstick testing.
a)Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) is a method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity. Unlike PCR, it doesn't require a sophisticated and extreme expensive equipment and a long time for result analysis. A small water bath kettle and no more than 40 minutes are adequate to proceed the whole testing.
b)Cas12a Protein and Lateral Flow testing
i)CRISPR Testing
Cas13a was discovered to possess the target RNA triggered 'collateral cleavage' activity in 2016. Gootenberg and coworkers developed CRISPR/Cas13a-based nucleic acid detection system, called SHERLOCK (specific high sensitivity enzymatic reporter unlocking). Soon after, the class II type V-A Cas12a (previously called Cpf1) effector was also found to possess collateral cleavage activity (or trans-cleavage activity). However, different from Cas13 effectors, Cas12a effectors target DNA and trans-cleave collateral ssDNA. The first report of the Cas12a trans-cleavage activity, as well as its usage in nucleic acid detection (one-hour low cost multipurpose highly efficient system or HOLMES) to detect DNA/RNA virus, single nucleotide polymorphism (SNP), etc.
Using a quenched fluorescent ssDNA reporter is able to detect any target DNA/RNA sequence. Also, it can be designed by lateral flow detection which much more convenient for field operation.
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