African swine fever virus detection chip and application thereof

Abstract

The invention relates to an African swine fever virus detection chip and application thereof.The detection chip comprises a plurality of transistors connected in parallel, each transistor comprises a substrate layer, an air connection gate electrode, a metal connection channel, an extension gate electrode and a biological detection layer, and the biological detection layer is formed by combining a biological detection chip with a biological probe through a surface modification technology; the sample to be detected is subjected to African swine fever virus detection.

Description

Technical Field

[0001] This invention relates to biochips and their applications, and particularly to biochips, bioprobes, and detection methods for detecting African swine fever virus. Background Technology

[0002] African swine fever virus (ASFV) is a double-stranded DNA virus that is highly contagious and has an extremely high mortality rate. It primarily affects pigs and does not infect humans. Initially discovered in Africa, ASF has spread worldwide due to the expansion of global trade and transportation. The virus has caused severe economic losses to the global pig farming industry. Since there is currently no effective vaccine or treatment, prevention focuses on detecting the virus and culling infected pigs.

[0003] Traditional virus detection methods, such as polymerase chain reaction (PCR), are difficult to perform outside of a laboratory and require specialized equipment and personnel. This makes rapid and effective detection on pig farms impossible, often delaying disease control. Furthermore, PCR is less sensitive; if the viral load is low, it may be difficult to accurately detect infected pigs, and PCR also suffers from nonspecificity.

[0004] In summary, how to effectively detect African swine fever virus and improve its shortcomings such as low sensitivity, long detection time, and complex operation is an urgent problem to be solved in this field. Summary of the Invention

[0005] In one aspect, the present invention provides a biosensor chip comprising a plurality of transistors arranged in an array, the array comprising a plurality of rows and a plurality of columns, each of the transistors being disposed in one of the plurality of columns and one of the plurality of rows, each of the transistors comprising:

[0006] A base layer includes a shared source, a shared drain, and a channel region disposed between the shared source and the shared drain;

[0007] An unconnected gate is disposed in the channel region, and the unconnected gate includes a polysilicon oxide layer;

[0008] A metal connection channel is provided on the unconnected gate;

[0009] An extended gate is disposed on the metal connection channel, the extended gate being electrically connected to the unconnected gate through the metal connection channel; and

[0010] A biodetection layer is disposed on the extended gate, and the biodetection layer includes a plurality of bioprobes, the bioprobes having a nucleotide sequence as shown in SEQ ID NO:1;

[0011] The biodetection layer of the plurality of transistors forms a biodetection area on the surface of a biodetection chip.

[0012] In some specific embodiments, the biodetection layer is obtained on the surface of the extended gate by a surface modification process.

[0013] In some specific embodiments, the biodetection area includes a microchannel or a sample tank.

[0014] On the other hand, the present invention provides a method for detecting African swine fever virus, which utilizes the above-mentioned biodetection chip to perform biological detection on a sample to be tested, the method comprising:

[0015] Provide one of the above-mentioned biological detection chips;

[0016] A blank sample is placed in the biological detection area, and a gate voltage and a drain current of the plurality of transistors are measured to establish a standard line.

[0017] A sample to be tested is placed in the biological detection area and left for a predetermined time.

[0018] The biodetection area is rinsed with the blank sample; and

[0019] The gate voltage and drain current of the plurality of transistors are measured to establish a measurement line, and the measurement line is compared with the standard line to determine whether the sample to be tested includes African swine fever virus.

[0020] In some specific embodiments, the sample to be tested is derived from a pig.

[0021] In some specific embodiments, the sample to be tested is a solution sample or a swab sample.

[0022] In some embodiments, the solution sample includes a whole blood sample, a serum sample, a nasal fluid sample, an oral fluid sample, or a urine sample. In some embodiments, the swab sample includes a nasal swab sample, an oral swab sample, or a rectal swab sample.

[0023] In another aspect, the present invention provides a biological probe composed of a nucleotide sequence as shown in SEQ ID NO:1, which is used to detect African swine fever virus.

[0024] In some specific embodiments, the biological probe is composed of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1.

[0025] The beneficial effects of the African swine fever virus detection chip of the present invention and its application are as follows:

[0026] The African swine fever detection chip provided by this invention has advantages such as high sensitivity, good accuracy and simple operation. In some specific embodiments, the African swine fever detection chip provided by this invention can be configured as a handheld detector. By connecting to a mobile phone or computer, data can be transmitted, analyzed and returned. At the same time, the biochip described in this invention can also detect various different test samples. It is quite simple to operate and can be directly tested in pig farms, which is helpful for industrial application.

[0027] Furthermore, the biological probe designed in this invention can detect different African swine fever variants while maintaining considerable sensitivity and accuracy, effectively promoting its application in industry. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of the biological detection chip described in this invention.

[0029] Figure 2 In one specific embodiment of the present invention, along Figure 1 A cross-sectional schematic diagram of the biodetection chip along the dashed line A-A'.

[0030] Figure 3 This is a schematic diagram of a surface modification process according to a specific embodiment of the present invention.

[0031] Figure 4 This is a signal quantification diagram of the biological detection chip described in Embodiment 2 of the present invention for the detection of African swine fever virus (ASFV).

[0032] Figure 5 This is a survival rate graph of pigs in each group according to Example 3 of the present invention.

[0033] Figure 6A This is a graph showing the changes in body temperature of pigs belonging to the low-virulence virus strain group.

[0034] Figure 6B This is a graph showing the changes in body temperature of pigs belonging to highly virulent virus strains.

[0035] Figure 6C This is a graph showing the changes in body temperature of pigs belonging to different recombinant virus strains.

[0036] Figure 7A This is a scatter plot showing the number of days since the virus was first detected in blood samples from low-virulence viral strains after challenge.

[0037] Figure 7B This is a scatter plot showing the number of days since the virus was first detected in blood samples from highly virulent viral strains after challenge.

[0038] Figure 7C This is a scatter plot showing the number of days after the first detection of the virus in blood samples from recombinant viral strain groups following challenge.

[0039] Figure 8A This is a scatter plot of the number of days after challenge when the virus was first detected in nasal swab samples from low-virulence virus strains.

[0040] Figure 8B This is a scatter plot of the number of days after the virus was first detected in nasal swab samples from highly virulent viral strains following challenge.

[0041] Figure 8C This is a scatter plot showing the number of days after the first detection of the virus in nasal swab samples from recombinant viral strain groups following challenge.

[0042] Figure 9A This is a scatter plot of the number of days after challenge when the virus was first detected in oral fluid samples from low-virulence virus strain groups.

[0043] Figure 9B This is a scatter plot of the number of days after the first detection of the virus in oral fluid samples from highly virulent viral strains following challenge.

[0044] Figure 9C This is a scatter plot showing the number of days after the first detection of the virus in oral fluid samples from recombinant viral strain groups following challenge.

[0045] Figure 10A This is a scatter plot of the number of days after challenge when the virus was first detected in rectal swab samples from low-virulence viral strains.

[0046] Figure 10B This is a scatter plot of the number of days after challenge for the first detection of the virus in rectal swab samples from highly virulent viral strains.

[0047] Figure 10C This is a scatter plot showing the number of days after the first detection of the virus in rectal swab samples from recombinant viral strain groups following challenge.

[0048] In the diagram: 1. Biodetection chip; 10. Substrate layer; 101. Source region; 102. Drain region; 103. Channel region; BP. Bioprobe; BA. Biodetection region; BL. Biodetection layer; D. Shared drain; EG. Extended gate; EGS. Extended gate surface; FG. Open gate; LL. Lower layer; MT. Metal connection channel; PL. Polysilicon oxide layer; S. Shared source; TA. Sample cell; T1-Tn. Transistor; UL. Upper layer. Detailed Implementation

[0049] The following examples illustrate embodiments of the present invention. Those skilled in the art can easily understand the advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different embodiments. Various details in this specification can also be modified and changed based on different viewpoints and applications without departing from the scope disclosed in the present invention.

[0050] In view of the shortcomings of African swine fever virus detection technology, this invention introduces a biochip into the detection of African swine fever and provides a biological probe with high accuracy and sensitivity to optimize the detection effect and operation process. The biochip provided by this invention generates energy changes through the interaction between the identification component and the analyte, and converts the energy changes into signals for subsequent detection and analysis. It has the effects of rapid detection, convenient operation and high sensitivity.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains, and are used in the context to describe the invention. The terminology used in this specification is for describing particular embodiments only and is not intended to limit the invention.

[0052] It should be noted that, as used herein, the singular terms “a,” “an,” and “the” include multiple indicators unless explicitly limited to one. Unless the context clearly indicates otherwise, the term “or” is used interchangeably with the term “and / or.”

[0053] As used herein, the terms “about,” “approximately,” or “approximately” substantially mean that the stated value or range is within 5%, preferably within 3%, and more preferably within 1%. The digitized quantities provided in this invention are approximate values, intended to be inferred even if the terms “about,” “approximately,” or “approximately” are not used.

[0054] As used herein, the term "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the term "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except trace impurities). The term "substantially consisting of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially alter the essential characteristics of such embodiments.

[0055] Unless otherwise defined herein, scientific and technical terms used in connection with this document should have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms should include plural, and plural terms should include singular. The methods and techniques of this invention can generally be carried out according to conventional methods known in the art. Generally, the nomenclature used to connect the following techniques, as well as techniques in biochemistry, enzymology, molecular and cell biology, microbiology, biochips, genetics and protein and nucleic acid chemistry, and hybrid reactions, described herein are all known and frequently used in the art. Unless otherwise stated, the methods and techniques of this invention can generally be carried out according to conventional methods known in the art and are described in the various general and more specific references cited and discussed in this specification.

[0056] In one embodiment, the present invention provides a biological detection chip comprising a plurality of transistors arranged in an array, the array comprising a plurality of rows and a plurality of columns, each of the plurality of transistors being disposed in one of the plurality of columns and one of the plurality of rows, each of the transistors individually comprising:

[0057] A base layer includes a shared source, a shared drain, and a channel region disposed between the shared source and the shared drain;

[0058] An unconnected gate is disposed in the channel region, and the unconnected gate includes a polysilicon oxide layer;

[0059] A metal connection channel is provided on the unconnected gate;

[0060] An extended gate is disposed on the metal connection channel, the extended gate being electrically connected to the unconnected gate through the metal connection channel; and

[0061] A biodetection layer is disposed on the extended gate, the biodetection layer including a plurality of bioprobes, wherein the bioprobes have a nucleotide sequence as shown in SEQ ID NO:1;

[0062] The biodetection layer of the plurality of transistors forms a biodetection area on the surface of a biodetection chip.

[0063] In some specific embodiments, the biosensor layer is obtained on the surface of the extended gate through a surface modification process. The surface modification process includes APTES-GA modification, APTES-NHS modification, APTES-Biotin modification, or GPTMS modification.

[0064] In some specific embodiments, the biodetection zone includes a microchannel or a sample tank for loading the sample to be tested.

[0065] In another embodiment, the present invention provides a method for detecting African swine fever virus, which utilizes a biodetection chip as described in the present invention to perform biological detection on a sample to be tested, the method comprising:

[0066] Provide a biological detection chip as described in this invention;

[0067] A blank sample is placed in the biological detection area, and a gate voltage and a drain current of the plurality of transistors are measured to establish a standard line.

[0068] A sample to be tested is placed in the biological detection area and left for a predetermined time.

[0069] The biodetection area is rinsed with the blank sample; and

[0070] The gate voltage and drain current of the plurality of transistors are measured to establish a measurement line, and the measurement line is compared with the standard line to determine whether the sample to be tested includes African swine fever virus.

[0071] In some specific embodiments, the sample to be tested is derived from a pig.

[0072] In some embodiments, the sample to be tested is a solution sample or a swab sample. In some embodiments, the solution sample includes whole blood, serum, nasal fluid, oral fluid, or urine. In some embodiments, the swab sample includes a nasal swab, an oral swab, or a rectal swab.

[0073] In another embodiment, the present invention provides a biological probe composed of a nucleotide sequence as shown in SEQ ID NO:1, which is used for detecting African swine fever virus. The biological probe is a biodetection chip for African swine fever virus.

[0074] In some specific embodiments, the biological probe is composed of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0075] As used in this invention, the term "GPTMS modification method" refers to a surface modification process involving treatment with 3-epoxypropanetrimethoxysilane (GPTMS).

[0076] The term "approximate distance" as used in this invention refers to two distances that are equal, or a second distance that is approximately equal to a first distance, meaning the second distance is within ±10% of the first distance. For example, if the first distance is 1 cm, then the second distance that is approximately equal to the first distance is 1 cm or 0.9 to 1.1 cm.

[0077] To understand the technical features, content, advantages, and effects of the present invention, the present invention will now be described in detail below with reference to the accompanying drawings and embodiments. The drawings used are for illustrative purposes only and to assist in the description. They may not represent the actual proportions and precise configurations of the present invention after implementation. Therefore, the proportions and configurations of the accompanying drawings should not be used to interpret or limit the scope of the present invention in actual implementation.

[0078] Structure of biodetection chips

[0079] Figure 1 This is a schematic diagram of a bio-detection chip according to an embodiment of the present invention. As shown in the figure, the transistors of the bio-detection chip 1 can be arranged in an array, and multiple transistors located in the same column can be connected in parallel, including n parallel transistors T1 to Tn. The number of parallel transistors can be determined according to the needs of the bio-detection chip 1. The structure of these parallel transistors T1 to Tn includes an upper UL layer and a lower LL layer. In the lower LL layer, taking transistor T1 as an example, the structure of the substrate layer 10 includes a shared source S and a shared drain D, with a channel region between them. An unconnected gate FG is disposed in the channel region between the shared source S and the shared drain D. A polysilicon oxide layer PL is disposed below the unconnected gate FG. A metal connection channel MT is disposed at the X-shaped position of the unconnected gate FG, and the metal connection channel MT extends to the upper UL layer, such as... Figure 1 At the position corresponding to the X' box, an extension gate EG is set on the metal connection channel MT. Through the metal connection channel MT, the extension gate EG of the upper UL is electrically connected to the unconnected gate FG of the lower LL.

[0080] Furthermore, the unconnected gate FG of transistor T1 and the unconnected gate FG of the adjacent transistor T1' in the same row below can be close to the same shared drain D at approximately the same distance. The unconnected gate FG of the adjacent transistor T1' in the same row below and the unconnected gate FG of the adjacent transistor T1" in the same row below can be close to the same shared source S at approximately the same distance.

[0081] In this embodiment, the extended gate EG is a metal sheet electrode connected to the covering polysilicon oxide layer PL via a metal connection channel MT. This allows the extended gate EG of the upper UL to be electrically connected to the unconnected gate FG. In other embodiments, the extended gate EG can also be a metal electrode of other shapes, such as a spiral electrode. On the other hand, the n parallel transistors T1 to Tn can also be arranged in multiple columns to form multiple detection column structures. Between each detection column, adjacent detection columns can share adjacent shared source S or shared drain D. When measuring transistors T1 to Tn, since these transistors T1 to Tn are arranged in parallel, the shared source S can be connected to the ground terminal, and the shared drain D is connected to the current measurement terminal. By applying a voltage to the extended gate EG, the sum of the drain currents is measured, and this is used as detection data for biological detection judgment.

[0082] For biosensing, a biosensing layer BL is placed on the extended gate EG of these transistors T1 to Tn. The biosensing layer BL binds to a specific detection target, causing a change in the transistor's electrical properties. The presence of the corresponding detection target is determined by measuring the current. For details on the configuration of the biosensing layer BL, please refer to [link to documentation]. Figure 2 , it is Figure 1 The specific details of the cross-sectional diagram along the dashed line A-A' are shown. The biodetection chip 1 includes multiple detection columns, and the transistors in each detection column include an upper UL layer and a lower LL layer structure. The upper UL layer structure also includes a biodetection layer BL structure.

[0083] like Figure 2 As shown, taking transistor Tn as an example, it includes a shared source S and a shared drain D. An unconnected gate FG is provided in the channel region between them. The unconnected gate FG is electrically connected to the extended gate EG through a metal connection channel MT. For example... Figure 2 As shown, in a specific embodiment, the unconnected gate FG of transistor Tn and the unconnected gate FG of the adjacent transistor Tn' in the same row can overlap with at least a portion of the same shared drain D, and the unconnected gate FG of the adjacent transistor Tn' in the same row and the unconnected gate FG of another adjacent transistor Tn' in the same row can overlap with at least a portion of the same shared source S.

[0084] The biodetection layer BL is disposed on the extended gate EG, wherein multiple bioprobes BP are fixed on the surface of the extended gate EG through a surface modification process to facilitate subsequent biodetection.

[0085] The biological probe BP used in this invention is a nucleic acid probe, wherein the nucleic acid probe has a nucleotide sequence as shown in SEQ ID NO:1, and is used to detect African swine fever virus. It is immobilized on the biodetection layer BL through biografting. During biodetection, the charge generated by the binding of these biological probes BP with the sample to be tested affects the drain current signal of the transistor Tn, thereby confirming whether the sample to be tested contains African swine fever virus.

[0086] The biodetection chip 1 can be formed by multiple parallel transistors to form a detection array, thereby forming a biodetection area BA on the surface of the biodetection chip 1. To enable these biodetection areas BA to accommodate the test solution containing biological material, the biodetection area BA can be provided with a sample groove TA, including a accommodating region, so that the test sample can be accommodated within the biodetection area BA, and the test solution can cover the extended gate surface EGS of the biodetection chip 1, allowing the analyte to contact and bind with the bioprobe BP of the biodetection layer BL. In this embodiment, the accommodating space is in the form of a sample groove TA, but the invention is not limited to this. In other embodiments, the biodetection area can also utilize a microchannel structure to form the accommodating and detection space.

[0087] In summary, such as Figure 1 and Figure 2 As shown, the present invention provides a biological detection chip 1, which includes a plurality of transistors arranged in an array, the array including a plurality of rows and a plurality of columns, each of the plurality of transistors being disposed in one of the plurality of columns and one of the plurality of rows, each of the transistors Tn individually including:

[0088] A base layer 10 includes a shared source S, a shared drain D, and a channel region 103 disposed between the shared source S and the shared drain D;

[0089] An unconnected gate FG is disposed in the channel region 103, and the unconnected gate FG includes a polysilicon oxide layer PL.

[0090] A metal connection channel MT is disposed on the unconnected gate FG;

[0091] An extended gate EG is disposed on the metal connection channel MT, and the extended gate EG is electrically connected to the unconnected gate FG through the metal connection channel MT; and

[0092] A biodetection layer BL is disposed on the extended gate EG, and the biodetection layer BL includes a plurality of bioprobes BP, wherein the bioprobes BP have a nucleotide sequence as shown in SEQ ID NO:1.

[0093] In this process, the biodetection layer BL of the plurality of transistors Tn forms a biodetection area BA on the surface of a biodetection chip 1.

[0094] In the array, the plurality of transistors T1 to Tn located in the same column are connected in parallel. Among the plurality of transistors (Tn, Tn', Tn"...) located in the same row, the unconnected gates FG of each pair of adjacent transistors (e.g., Tn and Tn') are respectively close to the shared source S or the shared drain D between the two transistors at approximately the same distance, or respectively cover at least a portion of the shared source S or the shared drain D between the two transistors.

[0095] The present invention is further illustrated by the following embodiments, which should not be construed as further limiting in any way. The entire contents of all references cited in this invention (including references, approved patents, disclosed patent inventions, and patent inventions co-invented) are expressly incorporated herein by reference.

[0096] Example 1. Preparation of African Swine Fever Detection Chip

[0097] In this specific embodiment, a surface modification process using GPTMS is performed on the surface of the extended gate to immobilize multiple biological probes with nucleotide sequences as shown in SEQ ID NO:1 onto the biological detection layer.

[0098] Please see Figure 3 , Figure 3 This is a schematic diagram of the GPTMS modification method according to an embodiment of the present invention. The specific contents of the GPTMS modification method include: cleaning the extended gate surface EGS, and then applying oxygen plasma to modify the chip surface so that the extended gate surface EGS has hydroxyl groups (-OH); immersing the biodetection chip in 3-epoxypropanol trimethoxysilane (GPTMS) prepared in anhydrous ethanol, and then immersing it in anhydrous ethanol for ultrasonic oscillation cleaning, and baking it at 90-120°C after cleaning; subsequently performing a biografting reaction with a bioprobe (SEQ ID NO:1) containing amino groups (-NH2) to fix the multiple bioprobes to the biodetection layer, rinsing with ultrapure water, and then spraying dry with nitrogen; then filling and rinsing with a filling agent such as bovine serum albumin (BSA), and then spraying dry with nitrogen, which can be used for vacuum preservation or subsequent detection.

[0099] Example 2. Detection effect of African swine fever detection chip

[0100] This specific embodiment tests the African swine fever detection chip described in this invention to explore its detection limit (LoD) for African swine fever virus.

[0101] Detection methods

[0102] In this embodiment, the detection method of the biosensor chip includes steps 1 to 5. The detection process of this embodiment can also be found in the AttoSense FET Biosensor kit.

[0103] Step 1: Provide a biodetection chip as described in this invention, wherein the fabrication method of the biodetection chip is as described in Example 1. In this example, the biodetection chip of this invention is inserted into a handheld detector for convenient subsequent use.

[0104] Step 2: Place the blank sample in the biodetection area and measure the gate voltage and drain current of multiple transistors to establish a standard line. In this embodiment, 140 μL of blank solution is taken into the sample cell for calibration to establish the standard line. After completion, the liquid in the sample cell is removed.

[0105] Step 3: Place the sample to be tested in the biodetection area and allow it to stand for a predetermined time. Inject the sample to be tested into the biodetection area, for example, by placing it in the sample well, and allow it to stand for a period of time to allow the sample to bind to the bioprobes of the biodetection layer. The standing time varies depending on the sample to be tested. In this embodiment, 140 μL of the sample to be tested is placed in the sample well and reacted for 15 minutes.

[0106] Step 4: Rinse the biodetection area with blank sample. To ensure more effective binding of the test sample to the biological probe, the biodetection area can be rinsed after a predetermined time, and the test sample can be placed again. This process of rinsing and placing can be repeated multiple times to ensure the amount of test sample bound. In this embodiment, 140 μL of blank solution is added to the sample tank for rinsing twice, and then the liquid in the sample tank is removed.

[0107] Step 5: Measure the gate voltage and drain current of multiple transistors to establish a measurement line. Compare the measurement line with the standard line to determine whether the sample to be tested contains the target. After completing the above rinsing and placement procedures, apply different gate voltages to the extended gate EG again. Measure the output current I1~In of the shared drain D using a current meter. Convert the sum of the output currents I1~In into a digital signal using a voltage converter. Output the drain current information to establish the measurement line for the sample to be tested. Compare the difference between the measurement line and the standard line. Determine whether the sample to be tested contains African swine fever virus by the change in the difference. If there is no significant shift in the measurement line, it indicates that the solution to be tested does not contain African swine fever virus, meaning the biological probe has not bound to the sample and will not affect the charge of the chip gate. Conversely, if the shift exceeds a predetermined range, it is determined that the sample to be tested contains African swine fever virus.

[0108] In some specific embodiments, during electrical measurements, a voltage of 0V-2V (0, 0.03, 0.06…2V) or 1.5V-3V is applied from the sample slot, resulting in 63 voltage values. The measurement software records the voltage value transmitted from the sensing area to the semiconductor component at each voltage and provides 63 sets of values. These values ​​are then plotted to generate an electrical curve. Furthermore, the term "chip signal value" used in the diagram refers to the quantization of the signal. This quantization is calculated using a ladder formula, which is used to calculate the area between the standard curve and the sample curve. The calculation method is as follows: |((Target2+Target1)*1 / 2)-((Baseline2+Baseline1)*1 / 2))|

[0109] The formula is: +|((Target3+Target2)*1 / 2)-((Baseline3+Baseline2)*1 / 2))|+…+|((Target63+Target62)*1 / 2)-((Baseline63+Baseline62)*1 / 2))|. Here, the chip signal value measured for the blank sample is considered the background signal value. If the chip signal value of the sample to be tested exceeds two standard deviations of the background signal value, the sample is determined to contain African swine fever virus and is considered a positive sample.

[0110] Experimental materials

[0111] The sample used in this embodiment was African swine fever virus particles. The sample was lysed with VLL buffer (LabTurbo™ Virus Mini Kit, model: LVN480-200, Letebo Biotechnology Co., Ltd., Taipei) for 15 minutes. The prepared sample was further diluted with 25 mM Tris-HCl buffer. After dilution, the virus concentrations of the test samples were 0 copies / μL (control group), 10 copies / μM, 10 copies / μL, and 10000 copies / μL, respectively, to test the detection limit of the biodetection chip described in this invention for African swine fever virus.

[0112] Experimental results

[0113] Please see Figure 4In the control group with a virus concentration of 0 copies / μL, a chip signal value of approximately 0.45 was detected, which was considered as the background signal value. The chip signal values ​​detected at virus concentrations of 10 copies / mL, 10 copies / μL, and 10000 copies / μL were 0.96, 2.33, and 4.56, respectively, showing that the higher the virus concentration, the stronger the chip signal. This confirms that the biological detection chip of the present invention does indeed have the ability to detect African swine fever virus, and that a virus concentration of 10 copies / mL can detect a value higher than the background signal, indicating that the detection limit of the biological chip of the present invention can detect a virus concentration of at least 10 copies / mL. Therefore, the biological detection chip of the present invention has high sensitivity.

[0114] Example 3. Animal experiments on African swine fever detection chips

[0115] In this embodiment, different African swine fever virus strains were challenged to pigs, and different test samples (e.g., blood samples or rectal swab samples) were taken to test the detection capability of the African swine fever detection chip described in this invention, and at the same time compared with the real-time PCR detection method.

[0116] Materials and Methods

[0117] Pigs were divided into a challenge group and a contact group. The challenge group was challenged with a low-virulence virus strain (ASFV p72 genotype I), a high-virulence virus strain (ASFV p72 genotype II), or a recombinant virus strain (ASFV p72 genotype I + ASFV p72 genotype II), respectively. The concentration of African swine fever virus was 10. 3 HAD 50 A total of 1 mL was used. Pigs in the contact group were not challenged; they were housed in the same area as the challenge group to observe the transmission of African swine fever virus. Low-virulence virus strains were challenged via intramuscular injection; high-virulence virus strains or recombinant virus strains were challenged via intranasal injection. The experimental design and number of pigs for each group are shown in Table 1 below.

[0118] Table 1. Experimental group design (number of pigs)

[0119] Virus attack team Contact group (no virus challenge) Low-virulence virus strains 3 3 Highly virulent virus strains 3 3 Recombinant virus strain 3 3

[0120] Following the challenge experiment, the body temperature of each pig was measured daily, and oral fluid, rectal swabs, and nasal swabs were collected from each pig. Blood samples were drawn from each pig every two days, and the above sample collection continued for 14 days. Each collected sample was tested using the African swine fever detection chip described in this invention, and simultaneously tested using real-time qPCR. In this embodiment, the number of days it took for each sample to first test positive was recorded, and the average value of samples with the same virus strain was taken to analyze on which day after challenge the African swine fever virus could be detected, thereby comparing the differences in effectiveness and sensitivity between chip detection and qPCR detection, and observing the viral infection status.

[0121] The samples collected in this embodiment were processed using the AttoSense FET Biosensor kit. Each collected sample underwent pretreatment: For solution samples: 20 μL of sample (whole blood, serum, oral fluid, or nasal fluid, etc.) was mixed with 80 μL of lysis buffer and allowed to stand for 10 minutes. For swab samples: a swab (taken from the nasal cavity, mouth, or stool) was inserted into 160 μL of lysis buffer, rotated 10 times to mix, and allowed to stand for 10 minutes. Subsequently, 20 μL of the pretreated sample was mixed with diluent for later use.

[0122] This embodiment uses quantitative real-time polymerase chain reaction (qPCR) for detection. The nucleotide sequence of the forward primer for the qPCR reaction is shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3. The probe has fluorescein amidite (FAM) attached to its 5' end and tetramethylrhodamine (TAM) attached to its 3' end. The probe (nucleotide sequence as shown in SEQ ID NO:4) and reaction solution consist of: 5 μL of 4X TaqMan Fast Virus 1-Step Master Mix (purchased from Biosystems). TM The reaction solution consisted of 10 μM primer pairs, 10 μM probe, and 5 μL of test solution, for a total of 20 μL. The optimal real-time qPCR reaction conditions were 50°C for 5 minutes, 95°C for 20 seconds, followed by 40 cycles of amplification (3 seconds at 95°C, 30 seconds at 58°C). All real-time qPCR reactions were performed using LightCycle. TM The data was completed on a 96 (purchased from Roche, Switzerland) and analyzed using its software.

[0123] Please refer to Embodiment 2 for the chip detection method in this embodiment. In this embodiment, the biological detection chip of the present invention is installed in a handheld detector and can be directly connected to a mobile phone or computer to quickly transmit detection data to the analysis device.

[0124] Experimental results

[0125] Please see Figure 5 This refers to the survival status of pigs in this embodiment over 14 days. The survival rates of pigs challenged with both the highly virulent virus strain and the recombinant virus strain were low. In particular, all pigs in the highly virulent virus strain challenge group died by day 9. Furthermore, pigs in the highly virulent virus strain contact group and the recombinant virus strain contact group also died in the later stages of the experiment. This indicates that unchallenged pigs were infected with African swine fever virus after contact with infected pigs, leading to their death. Please also refer to... Figure 6A , 6B The chart for 6C shows the average daily body temperature of pigs in each group. When a pig's body temperature exceeds 40°C, it indicates that a fever has occurred. The results showed that the low-virulence virus strain challenge group, the high-virulence virus strain challenge group, and the recombinant virus strain challenge group developed fever around days 4-6. The contact group also developed fever. In particular, the high-virulence virus strain contact group and the recombinant virus strain contact group showed that after day 10, only the unchallenged pigs were infected.

[0126] A. Blood sample testing

[0127] Please see Figure 7A Using the chip described in this invention, African swine fever virus (ASFV) was detected in blood samples from the low-virulence virus strain challenge group on days 2, 4, and 4, respectively, while ASFV was detected in blood samples from the low-virulence virus strain contact group on days 1, 4, and 8, respectively. On average, a positive reaction could be detected by the chip on day 3.83 post-challenge. Using qPCR, ASFV was detected in blood samples from the low-virulence virus strain challenge group on days 4, 4, and 4, respectively, while ASFV was detected in blood samples from the low-virulence virus strain contact group on days 14, 14, and 14, respectively. On average, a positive reaction was detected on day 9 post-challenge.

[0128] Please see Figure 7BUsing the chip described in this invention, African swine fever virus (ASFV) was detected in blood samples from the highly virulent virus strain challenge group on days 2, 2, and 4, respectively, while ASFV was detected in blood samples from the highly virulent virus strain contact group on days 2, 4, and 6, respectively. On average, a positive reaction could be detected by the chip on day 3.33 post-challenge. Using qPCR, ASFV was detected in blood samples from the highly virulent virus strain challenge group on days 4, 4, and 4, respectively, while ASFV was detected in blood samples from the highly virulent virus strain contact group on days 6, 10, and 14, respectively. On average, a positive reaction was detected on day 7 post-challenge.

[0129] Please see Figure 7C Using the chip described in this invention, African swine fever virus (ASFV) was detected in blood samples from the recombinant virus challenge group on days 2, 2, and 10, respectively, while ASFV was detected in blood samples from the recombinant virus contact group on days 6, 8, and 8, respectively. On average, a positive reaction could be detected by the chip on day 6 post-challenge. Using qPCR, ASFV was detected in blood samples from the recombinant virus challenge group on days 4, 4, and 10, respectively, while ASFV was detected in blood samples from the recombinant virus contact group on days 10, 10, and 14, respectively. On average, a positive reaction was detected 8.67 days post-challenge.

[0130] The test results of blood samples on each day are shown in Tables 2-1 to 2-3 below. In the tables, the denominator represents the total number of pigs, and the numerator represents the number of pigs that tested positive. A positive result indicates that African swine fever virus was detected in the sample from that pig. For example, 0 / 3 means that out of 3 pigs, 0 tested positive; 1 / 3 means that out of 3 pigs, 1 tested positive; 2 / 3 means that out of 3 pigs, 2 tested positive; and 3 / 3 means that out of 3 pigs, 3 tested positive. Additionally, samples from dead pigs are considered positive samples.

[0131] The blood samples of low-virulence virus strains were tested as shown in Table 2 below. When the contact group of low-virulence virus strains was tested by real-time PCR, African swine fever virus could not be detected. This is because the virus concentration was low. In contrast, the infection status of pigs could be detected by chip detection, which shows that the African swine fever detection chip of the present invention has higher sensitivity.

[0132] Table 2. Blood Sample Testing - Low Virus Strains

[0133]

[0134]

[0135] The results of blood sample testing for highly virulent virus strains are shown in Table 3 below. The results show that the chip detection is more effective than PCR detection, especially in the contact group of highly virulent virus strains. The African swine fever detection chip described in this invention has higher sensitivity.

[0136] Table 3. Blood Sample Testing - Highly Viral Virus Strains

[0137]

[0138] The blood sample tests of the recombinant virus strain are shown in Table 4 below. The results show that the chip detection is more effective than PCR detection, especially in the recombinant virus strain contact group, where the African swine fever detection chip described in this invention exhibits higher sensitivity.

[0139] Table 4. Blood Sample Detection - Recombinant Virus Strain

[0140]

[0141]

[0142] Based on the test results of blood samples, the African swine fever detection chip described in this invention can detect African swine fever virus in pigs on average only 4.386 days from the date of infection. In contrast, qPCR detection requires a higher viral concentration in the pig to be effective, averaging 8.223 days. Furthermore, the stability of the chip detection can be observed through continuous monitoring. The results show that the African swine fever detection chip described in this invention can be used for blood samples, has higher sensitivity, and is suitable for detecting different African swine fever virus variants.

[0143] B. Nasal swab sample testing

[0144] Please see Figure 8AUsing the chip described in this invention, African swine fever virus (ASFV) was detected in nasal swab samples from the low-virulence virus strain challenge group on days 4, 4, and 5, respectively, while ASFV was detected in nasal swab samples from the low-virulence virus strain contact group on days 4, 5, and 7, respectively. On average, a positive reaction could be detected by the chip on day 4.83 post-challenge. Using qPCR, ASFV was detected in nasal swab samples from the low-virulence virus strain challenge group on days 5, 6, and 7, respectively, while ASFV was detected in nasal swab samples from the low-virulence virus strain contact group on days 8, 13, and 14, respectively. On average, a positive reaction was detected on day 8.83 post-challenge. The results show that ASFV was difficult to detect in the low-virulence virus strain contact group using real-time qPCR, due to the low viral concentration, requiring approximately day 8 for a more detectable result. In contrast, chip detection effectively detected infection in pigs even in the early stages when viral load was low, demonstrating the higher sensitivity of the ASFV detection chip described in this invention.

[0145] Please see Figure 8B Using the chip described in this invention, African swine fever virus (ASFV) was detected in nasal swab samples from the highly virulent virus strain challenge group on days 2, 2, and 4, respectively. In contrast, ASFV was detected in nasal swab samples from the highly virulent virus strain contact group on days 4, 6, and 7, respectively. On average, a positive reaction was detectable by the chip on day 4.17 post-challenge. Using qPCR, ASFV was detected in nasal swab samples from the highly virulent virus strain challenge group on days 4, 4, and 5, respectively. In contrast, ASFV was detected in nasal swab samples from the highly virulent virus strain contact group on days 10, 10, and 12, respectively. On average, a positive reaction was detected on day 7.5 post-challenge. The results show that the chip detection is more effective than qPCR detection, especially in the highly virulent virus strain contact group. qPCR detection only becomes effective in the later stages when the virus concentration is high, while chip detection detects the virus in the early stages, demonstrating the higher sensitivity of the ASFV detection chip described in this invention.

[0146] Please see Figure 8CUsing the chip described in this invention, African swine fever virus (ASFV) was detected in nasal swab samples from the recombinant virus strain challenge group on days 2, 3, and 4, respectively, while ASFV was detected in nasal swab samples from the recombinant virus strain contact group on days 2, 6, and 7, respectively. On average, a positive reaction could be detected by the chip on day 4 after challenge. Using qPCR, ASFV was detected in nasal swab samples from the recombinant virus strain challenge group on days 5, 5, and 11, respectively, while ASFV was detected in nasal swab samples from the recombinant virus strain contact group on days 10, 11, and 13, respectively. On average, a positive reaction was detected on day 9.17 after challenge. The results show that chip detection is effective even when the virus concentration is low in the early stages of the experiment, while qPCR detection struggles to detect low concentrations of virus. This demonstrates that chip detection is superior to qPCR detection, especially in the recombinant virus strain contact group, where the ASFV detection chip described in this invention exhibits higher sensitivity.

[0147] Overall, the results of nasal swab sample testing show that using the African swine fever detection chip described in this invention, the virus can be detected in pigs on average only 4.33 days from the date of infection. However, qPCR testing requires a higher viral concentration in the pig to be effective, taking an average of 8.5 days, and necessitates bringing the sample back to the laboratory for a real-time qPCR reaction, which is more cumbersome. The results demonstrate that the African swine fever detection chip described in this invention can be used for nasal swab samples with higher sensitivity and is suitable for detecting different African swine fever virus variants.

[0148] C. Oral fluid sample testing

[0149] Please see Figure 9A Using the chip described in this invention, African swine fever virus (ASFV) was detected in oral fluid samples from the low-virulence virus strain challenge group on days 1, 3, and 5, respectively, while ASFV was detected in oral fluid samples from the low-virulence virus strain contact group on days 2, 3, and 4, respectively. On average, a positive reaction could be detected by the chip on day 3 after challenge. Using qPCR, ASFV was detected in oral fluid samples from the low-virulence virus strain challenge group on days 6, 7, and 10, respectively, while ASFV was detected in oral fluid samples from the low-virulence virus strain contact group on days 8, 10, and 11, respectively. On average, a positive reaction was detected 8.67 days after challenge. The results show that ASFV was difficult to detect in the low-virulence virus strain contact group using real-time qPCR, due to the low viral concentration, requiring approximately day 9 for a more detectable result. In contrast, chip detection effectively detected infection in pigs even in the early stages when viral load was low, demonstrating the higher sensitivity of the ASFV detection chip described in this invention.

[0150] Please see Figure 9B Using the chip described in this invention, African swine fever virus (ASFV) was detected in oral fluid samples from the highly virulent virus strain challenge group on days 2, 2, and 3, respectively, while ASFV was detected in oral fluid samples from the highly virulent virus strain contact group on days 5, 5, and 5, respectively. On average, a positive reaction could be detected by the chip on day 3.6 post-challenge. Using qPCR, ASFV was detected in oral fluid samples from the highly virulent virus strain challenge group on days 6, 6, and 6, respectively, while ASFV was detected in oral fluid samples from the highly virulent virus strain contact group on days 8, 10, and 14, respectively. On average, a positive reaction was detected on day 8.3 post-challenge. The results show that the chip detection is more effective than qPCR detection. In both the highly virulent virus strain challenge group and the contact group, qPCR detection only became effective when the virus concentration was high in the later stages, while chip detection could detect the virus in the early stages, demonstrating that the ASFV detection chip described in this invention has higher sensitivity.

[0151] Please see Figure 9C Using the chip described in this invention, African swine fever virus (ASFV) was detected in oral fluid samples from the recombinant virus challenge group on days 1, 3, and 4, respectively, while ASFV was detected in oral fluid samples from the recombinant virus contact group on days 2, 3, and 6, respectively. On average, a positive reaction could be detected by the chip on day 3.17 post-challenge. Using qPCR, ASFV was detected in oral fluid samples from the recombinant virus challenge group on days 5, 5, and 12, respectively, while ASFV was detected in oral fluid samples from the recombinant virus contact group on days 10, 10, and 14, respectively. On average, a positive reaction was detected on day 9.33 post-challenge. The results show that chip detection is effective at detecting low viral loads in the early stages of the experiment, while qPCR detection struggles to detect low concentrations of the virus. This demonstrates that chip detection is superior to qPCR detection. The ASFV detection chip described in this invention exhibits higher sensitivity in both the recombinant virus challenge and contact groups.

[0152] Based on the test results of oral fluid samples, the African swine fever detection chip described in this invention can detect African swine fever virus in pigs on average only 3.256 days from the date of infection. In contrast, PCR detection requires a high viral concentration in the pig to be effective, taking an average of 8.766 days, and necessitates bringing the sample back to the laboratory for real-time qPCR, which is more cumbersome. The results show that the African swine fever detection chip described in this invention can be used for oral fluid samples, has higher sensitivity, and is suitable for detecting different African swine fever virus variants.

[0153] D. Rectal swab sample testing

[0154] Please see Figure 10A Using the chip described in this invention, African swine fever virus (ASFV) was detected in rectal swab samples from the low-virulence virus strain challenge group on days 3, 3, and 7, respectively. In contrast, ASFV was detected in rectal swab samples from the low-virulence virus strain contact group on days 3, 4, and 4, respectively. On average, a positive reaction was detectable by the chip on day 4 post-challenge. Using qPCR, ASFV was detected in rectal swab samples from the low-virulence virus strain challenge group on days 4, 6, and 14, respectively. In contrast, ASFV was detected in rectal swab samples from the low-virulence virus strain contact group on days 14, 14, and 14, respectively. On average, a positive reaction was detected on day 11 post-challenge. The results show that ASFV could not be detected by real-time qPCR in the low-virulence virus strain contact group due to the low viral concentration. In comparison, chip detection effectively detected the infection status of pigs even in the early stages when the viral load was low, demonstrating the higher sensitivity of the ASFV detection chip described in this invention.

[0155] Please see Figure 10B Using the chip described in this invention, African swine fever virus (ASFV) was detected in rectal swab samples from the highly virulent virus strain challenge group on days 2, 3, and 5, respectively. In contrast, ASFV was detected in rectal swab samples from the highly virulent virus strain contact group on days 3, 4, and 5, respectively. On average, a positive reaction could be detected by the chip on day 3.67 post-challenge. Using qPCR, ASFV was detected in rectal swab samples from the highly virulent virus strain challenge group on days 4, 6, and 6, respectively. In contrast, ASFV was detected in rectal swab samples from the highly virulent virus strain contact group on days 11, 11, and 13, respectively. On average, a positive reaction was detected on day 8.5 post-challenge. The results show that the chip detection is more effective than qPCR detection, especially in the highly virulent virus strain contact group. qPCR detection only became effective in the later stages when the virus concentration was high, while chip detection detected the virus in the early stages, demonstrating the higher sensitivity of the ASFV detection chip described in this invention.

[0156] Please see Figure 10CUsing the chip described in this invention, African swine fever virus (ASFV) was detected in rectal swab samples from the recombinant virus challenge group on days 3, 3, and 4, respectively, while ASFV was detected in rectal swab samples from the recombinant virus contact group on days 5, 5, and 6, respectively. On average, a positive reaction could be detected by the chip on day 4.33 post-challenge. Using qPCR, ASFV was detected in rectal swab samples from the recombinant virus challenge group on days 5, 5, and 11, respectively, while ASFV was detected in rectal swab samples from the recombinant virus contact group on days 10, 11, and 14, respectively. On average, a positive reaction was detected on day 9.33 post-challenge. The results show that chip detection is effective at detecting low viral loads in the early stages of the experiment, while qPCR detection struggles to detect low concentrations of the virus. This demonstrates that chip detection is superior to qPCR detection. The ASFV detection chip described in this invention exhibits higher sensitivity in both the recombinant virus challenge and contact groups.

[0157] Based on the results of rectal swab sample testing, the African swine fever detection chip described in this invention can detect the African swine fever virus in pigs on average only 4 days from the date of infection. However, qPCR testing requires a higher viral concentration in the pig to be effective, taking an average of 9.61 days, and necessitates bringing the sample back to the laboratory for real-time qPCR reaction, which is more cumbersome. The results show that the African swine fever detection chip described in this invention can be used for rectal swab samples with higher sensitivity and is suitable for detecting different African swine fever virus variants.

[0158] In summary, the African swine fever detection chip of this invention can effectively detect low levels of the virus, exhibiting high sensitivity and enabling rapid detection in the early stages of infection in pigs, thus facilitating epidemic control. It also demonstrates high detection stability. Furthermore, the African swine fever detection chip is applicable to various samples, and the biological probe described in this invention can effectively detect different variants of the African swine fever virus. The chip is simple and quick to operate, making it suitable for industrial applications and showing broad prospects for the prevention and control of African swine fever virus.

[0159] The above detailed description is a specific description of a feasible embodiment of the present invention. However, the embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the spirit of the present invention should be included in the patent scope of the present invention.

Claims

1. A biosensor chip comprising a plurality of transistors arranged in an array, the array comprising a plurality of rows and a plurality of columns, each of the transistors being disposed in one of the plurality of columns and one of the plurality of rows, each of the transistors comprising: A base layer includes a shared source, a shared drain, and a channel region disposed between the shared source and the shared drain; An unconnected gate is disposed in the channel region, and the unconnected gate includes a polysilicon oxide layer; A metal connection channel is provided on the unconnected gate; An extended gate is disposed on the metal connection channel, the extended gate being electrically connected to the unconnected gate through the metal connection channel; and A biodetection layer is disposed on the extended gate, the biodetection layer comprising a plurality of bioprobes, characterized in that the bioprobes have a nucleotide sequence as shown in SEQ ID NO:1; The biodetection layer of the plurality of transistors forms a biodetection area on the surface of a biodetection chip.

2. The biodetection chip according to claim 1, characterized in that, The biosensor layer is obtained on the surface of the extended gate through a surface modification process.

3. The biodetection chip according to claim 1, characterized in that, The biodetection area includes a microchannel or a sample tank.

4. A method for detecting African swine fever virus, characterized in that, The method for performing biological detection on a sample using the biodetection chip as described in claim 1 includes: Provide a biodetection chip as described in claim 1; A blank sample is placed in the biological detection area, and a gate voltage and a drain current of the plurality of transistors are measured to establish a standard line. A sample to be tested is placed in the biological detection area and left for a predetermined time. The biodetection area is rinsed with the blank sample; and The gate voltage and drain current of the plurality of transistors are measured to establish a measurement line, and the measurement line is compared with the standard line to determine whether the sample to be tested includes African swine fever virus.

5. The detection method according to claim 4, characterized in that, The sample to be tested came from a pig.

6. The detection method according to claim 4, characterized in that, The sample to be tested is either a solution sample or a swab sample.

7. The detection method according to claim 6, characterized in that, The solution samples include whole blood samples, serum samples, nasal fluid samples, oral fluid samples, or urine samples.

8. The detection method according to claim 6, characterized in that, The swab samples include nasal swab samples, oral swab samples, or rectal swab samples.

9. A biological probe, characterized in that, It consists of a nucleotide sequence as shown in SEQ ID NO:1 and is used to detect African swine fever virus.

10. The biological probe according to claim 9, characterized in that, It consists of a nucleotide sequence that has at least 90% sequence identity with SEQ ID NO:1.

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