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What is the core technology of the African swine fever rapid detection device? Why can viruses be accurately identified

Why can a detector detect a virus in just a few tens of minutes? "" What technical tricks are hidden behind the differences in precision between different devices? "The" golden eyes "of the African swine fever rapid detector stem from breakthroughs in three core technologies. These technologies achieve precise judgment from "presence or absence of virus" to "number of virus" by locking the "unique identifier" of the virus, which is a deep integration of molecular biology and engineering technology.

1、 Colloidal gold immunochromatography technology: a "grassroots sentinel" that produces results in 15 minutes

The colloidal gold rapid detector is a device for free range households, and its core technology is essentially the "specific binding of viral antigens and antibodies", similar to the precise matching of "key to lock".

There are two key lines hidden in the detection card of this type of device: the detection line (T line) fixes the specific antibody of African swine fever virus P72 protein, and the quality control line (C line) fixes the anti gold antibody. During detection, the virus antigen in the sample will bind to the colloidal gold labeled antibody, forming a "gold labeled antibody virus antigen" complex. When the complex flows to the T-line, it will be captured by the T-line antibody, and the aggregated colloidal gold will form a red band, indicating positivity; Unbound gold labeled antibodies will continue to flow to the C-line and be captured, forming a red band to confirm the effectiveness of the detection.

The precision of this technology comes from the "specificity" of antibodies. Antibodies targeting the P72 protein only bind to African swine fever virus and do not react with other viruses such as swine fever and blue ear disease. The specificity can reach over 95%. The experience of Mr. Li, a free range farmer in Shandong, confirms this point: "Using a colloidal gold card to detect febrile pigs, the results are obtained within 15 minutes, and subsequent laboratory review is consistent without any misjudgment." However, its sensitivity is limited, and it can only detect medium to high concentrations of viruses (≥ 1000 copies/μ L), which is more suitable for emergency screening.

2、 Real time fluorescence quantitative PCR technology: the "gold standard" for accurate diagnosis

PCR technology is the core of diagnosis and detection in small and medium-sized pig farms. Its core is the specific amplification and fluorescence tracking of viral nucleic acids, which can accurately capture the presence of single copy viruses.

The detection logic of such devices is divided into three steps: first, the virus shell is destroyed by lysis buffer to extract its unique DNA (targeting the highly conserved B646L gene, which is common to all African swine fever viruses); Subsequently, specific primers were added to lock onto the B646L gene fragment like a "localization anchor". Under the action of DNA polymerase, the target fragment was amplified millions of times through a "denaturation annealing extension" cycle reaction; At the same time, the fluorescent probe will release a fluorescent signal during the amplification process, and the device can calculate the initial viral load by tracking the signal intensity in real time.

The measured data of TH-P800 PCR instrument shows that it can detect viruses as low as 10 copies/μ L, which is equivalent to finding 1 salt particle in 1 ton of water. Zhao Lei, a veterinarian at a pig farm in Hebei Province, explained: "Even if the pigs have just been infected and the amount of virus in their blood is very small, after 30 cycles of amplification, they can still be captured by fluorescent signals, which is the key to early diagnosis." The specificity of this technology lies in the primer design targeting the B646L gene, which is specific to African swine fever virus and does not bind to genes of other viruses.

3、 RPA-CRISPR/Cas12a combined technology: a new generation of "precision Kwai"

As a new technology that has emerged in recent years, the RPA-CRISPR combination device achieves a balance between "speed and accuracy", with sensitivity even surpassing traditional PCR. The core is the dual verification of "isothermal amplification+gene editing tools".

The detection process is divided into two steps: using recombinant polymerase chain reaction (RPA) technology, the B646L gene of the virus is amplified to a detectable level within 15 minutes under constant temperature conditions of 37-42 ℃, solving the time-consuming problem of repeated temperature rise and fall required by traditional PCR; The second step is to activate the pre designed crRNA of the CRISPR/Cas12a system, which acts as a "guide" to locate the amplified viral gene and activate the cleavage activity of Cas12a, causing it to cleave nearby fluorescent reporter molecules and release fluorescent signals.

Research from Shenzhen Institute shows that this technology has a sensitivity of 20 copies/mL and can achieve colorimetric fluorescence dual-mode detection: positive samples will appear yellow while emitting fluorescent signals, and dual verification avoids misjudgment caused by environmental interference. In the detection of 12 real samples of pig blood, its accuracy reached 100% and it did not cross react with other pig disease viruses. Liu Feng, the veterinary director of a certain breeding group, commented: "Previously, PCR took 2 hours, but now RPA-CRISPR equipment can produce results in 40 minutes with higher accuracy, making it particularly suitable for batch screening. ”

4、 A common password for precise identification: a unique identifier for locking viruses

Regardless of the technology, the core of precise identification is to identify the "irreplaceable features" of African swine fever virus:

Target specificity: The highly conserved B646L gene of the virus is used as the detection target, which is present in all African swine fever virus strains with very little variation, ensuring no missed detection or misjudgment;

Signal amplification: By amplifying trace amounts of viral nucleic acid millions of times through PCR or RPA technology, and then converting signals through fluorescence, colloidal gold, etc., the "invisible virus" can be transformed into a "visible result";

Specific binding: The binding between antibodies and antigens, primers and genes is like a "fingerprint match", only reacting to African swine fever virus, eliminating false positives caused by cross contamination from the source.

A pig farm in Henan Province suffered losses exceeding 100000 yuan due to misjudgment caused by the use of unqualified equipment. It can be seen that the accuracy of equipment depends not only on the type of technology, but also closely related to target selection and reagent quality. For farmers, understanding these technical principles can help them more clearly determine whether the equipment is suitable for emergency screening needs, select colloidal gold for precise diagnosis using PCR, and focus on the new RPA CRISPR technology for efficient batch testing.