Chicken infectious anemia virus isothermal amplification rapid detection kit, method and application

Abstract

According to the chicken infectious anemia virus isothermal amplification rapid detection kit, the method and the application, the primers and the probes are designed and optimized, and high affinity and specific binding with target nucleic acid are ensured. The method has the beneficial effects of rapidness, convenience, high sensitivity and specificity, visual result interpretation, wide sample applicability, high cost effectiveness, good stability and reliability, capability of effectively avoiding dependence on large-scale instruments in laboratories and the like; the method has an important application value and a wide market prospect in the fields of detection, prevention and control of chicken infectious anemia.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, and relates to a rapid detection kit, method and application of chicken infectious anemia virus isothermal amplification. Background Technology

[0002] Chicken Infectious Anemia Virus (CIAV) is a major pathogen threatening the poultry industry. Infection in chickens leads to anemia, immunosuppression, and other symptoms, severely impacting growth, development, and productivity, causing significant economic losses for poultry farmers. This virus is highly contagious and can spread through multiple routes, such as vertical and horizontal transmission. Once an outbreak occurs in a flock, it is difficult to control. Therefore, rapid and accurate detection of CIAV is crucial for epidemic prevention and control and minimizing losses.

[0003] Currently, diagnostic techniques for infectious anemia in chickens primarily rely on clinical diagnosis, virus isolation and identification, enzyme-linked immunosorbent assay (ELISA), immunoenzyme assay, polymerase chain reaction (PCR), and fluorescent polymerase chain reaction (qPCR) assays, along with comprehensive judgment techniques (NY / T 1187-2019). However, conventional pathogen and serological detection methods are complex, time-consuming, and require stringent experimental conditions, making them unsuitable for rapid on-site testing. While polymerase chain reaction (PCR) and fluorescent polymerase chain reaction (qPCR) offer high sensitivity, they require complex equipment (such as PCR instruments and gel imaging systems), involve numerous steps including nucleic acid extraction, amplification, and electrophoresis, and are time-consuming, similarly making them unsuitable for rapid on-site testing. Furthermore, these detection methods are costly. Summary of the Invention

[0004] To address the above-mentioned technical problems, the present invention aims to provide a rapid detection kit, method, and application for isothermal amplification of chicken infectious anemia virus. This kit offers numerous advantages, including rapid and convenient detection, high sensitivity and specificity, visual result interpretation, wide sample applicability, cost-effectiveness, good stability and reliability, and effective elimination of reliance on large laboratory instruments. It has significant application value and broad market prospects in the detection and prevention of chicken infectious anemia.

[0005] To achieve its technical objectives, the present invention employs the following technical solution:

[0006] This invention provides primers and probes for rapid detection of chicken infectious anemia virus by isothermal amplification, wherein:

[0007] The nucleotide sequence of the upstream primer is as follows:

[0008] 5'-TGGTATCGCTGGAATTACAATCRCTCTATC-3', R=A / G, as shown in SEQ ID NO:1;

[0009] The nucleotide sequence of the downstream primer is as follows:

[0010] 5'-TTCTTCGAGGGAGGCTTGGSTTGATCGGTC-3', S=C / G, as shown in SEQ ID NO:8, the 5' end is modified with biotin;

[0011] The nucleotide sequence of the probe is as follows:

[0012] 5'-CGAATGCTCGCGCTCCCACGCTAAGATCTG[THF]AACTGCGGACAATTC-3', as shown in SEQ ID NO:6, has FAM modified at the 5' end, C3Spacer modified at the 3' end, and tetrahydrofuran (THF) modified at the 31bp midpoint of the 5' end.

[0013] This invention also provides a rapid detection kit for isothermal amplification of chicken infectious anemia virus, including the aforementioned primers and probes.

[0014] Preferably, the kit further includes an activator, a nucleic acid chromatography test strip, a positive control sample, and a negative control sample.

[0015] More preferably, the activator is 21mM magnesium acetate; the positive control sample is chicken infectious anemia virus nucleic acid, and the negative control sample is SPF chicken embryo grinding fluid nucleic acid.

[0016] This invention also provides the application of the above-mentioned primers and probes or the above-mentioned kits in the detection of chicken infectious anemia virus.

[0017] Preferably, the samples tested include live chicken samples, necropsy samples of dead chickens or necropsy samples from slaughterhouses, and environmental samples contaminated by diseased chickens; the isothermal amplification temperature is 42°C and the time is 15 minutes.

[0018] This invention also provides a rapid detection method for isothermal amplification of chicken infectious anemia virus, which is achieved using the above-mentioned primers and probes or the above-mentioned kit. The method includes: mixing the sample nucleic acid with a reaction solution containing primers and probes, then immediately placing the reaction tube in a 42°C isothermal device for 15 min; after the reaction is completed, diluting the reaction solution with ddH2O, and then dropping the diluted solution into the sample port of the nucleic acid chromatography test strip for chromatography detection; after standing for 10-15 min, observing the control line (C line) and the test line (T line) and interpreting the results.

[0019] Preferably, if a band appears on line C of the test strip, no band appears on line T of the negative control sample, and a red band appears on line T of the positive control sample, the test result is valid; if no band appears on line C of the test strip, regardless of whether a band appears on line T, the result is invalid and needs to be tested again.

[0020] Under the conditions that the test is valid, if the test strip C line shows a red band and the T line shows no band, it is judged as negative for chicken infectious anemia virus nucleic acid; if the test strip C line shows a red band and the T line shows a red band, the color of which can be dark or light, it is judged as positive for chicken infectious anemia virus nucleic acid.

[0021] Preferably, the reaction is carried out according to the following reaction system:

[0022]

[0023] .

[0024] Preferably, the method for obtaining nucleic acid from the sample includes:

[0025] i. Live chicken samples: Aseptically collect 5 mL of anticoagulated blood or serum from live chickens, and then take 200 μL of the sample for nucleic acid extraction;

[0026] ii. Necropsy samples from dead chickens or slaughterhouse samples: Aseptically collect tissue samples (liver, spleen, etc.) from dead chickens, and use sterile scissors and tweezers to cut the sample to be tested (about 0.5 g, it is better to cut it into small pieces) into a grinding tube for nucleic acid extraction;

[0027] iii. Environmental samples contaminated by infected chickens: Feces from places associated with infected chickens are placed in a one-step virus release solution to prepare a homogenate of about 10% for nucleic acid extraction; or 200 μL of sewage is taken directly for nucleic acid extraction.

[0028] The beneficial effects of this invention are as follows:

[0029] I. Fast and convenient testing process

[0030] This invention offers a simple and rapid process from sample collection and processing to nucleic acid extraction, isothermal amplification, and lateral flow detection, greatly simplifying the detection procedure. For example, for tissue samples such as liver and spleen, only about 0.5 g needs to be cut and placed in a grinding tube for nucleic acid extraction; for samples such as whole blood and serum, 200 μL can be directly extracted. This simplified sample processing method saves significant time and labor costs compared to traditional, complex nucleic acid extraction methods. Moreover, the isothermal amplification reaction only needs to be carried out in a 42°C isothermal device for 15 minutes, followed by detection using nucleic acid chromatography test strips, with results visible within 10-15 minutes. The entire detection process can be completed in a short time, providing strong support for the rapid diagnosis of avian infectious anemia virus, and is particularly suitable for rapid on-site detection and widespread application in grassroots laboratories.

[0031] II. Detection Results with High Sensitivity and Specificity

[0032] This invention utilizes carefully designed primers and probes to specifically identify the nucleic acid sequence of chicken infectious anemia virus (TIV). The nucleotide sequences of the primers and probes are optimized to ensure high affinity and specific binding to the target nucleic acid. During isothermal amplification, the target nucleic acid is amplified efficiently, accurately detecting the presence of TIV even in samples with low viral loads. This high sensitivity and specificity facilitates early detection of infected chickens, enabling timely implementation of control measures and reducing the risk of virus transmission, which is of great significance for the prevention and control of TIV.

[0033] III. Interpretation of Visual Results

[0034] This invention utilizes nucleic acid chromatography test strips for result interpretation, offering a direct and objective approach. The control line (C line) and test line (T line) on the test strip are clearly visible, eliminating the need for complex instruments and specialized personnel for result analysis. Under valid test conditions, a red band on the C line indicates a valid test, while the presence and intensity of the T line directly reflect the positive or negative result of the sample. This visual result interpretation method reduces human error, improves the accuracy and reliability of test results, and allows even non-professionals to easily master the testing method, facilitating its widespread application in practical production.

[0035] IV. Wide sample applicability

[0036] This invention is applicable to various types of samples, including anticoagulated blood or serum from live chickens, tissue samples such as the liver and spleen from dead chickens, and environmental samples such as feces and sewage associated with diseased chickens. This broad sample applicability makes the detection range more comprehensive, enabling monitoring of the presence and spread of avian infectious anemia virus from different perspectives. Whether for health monitoring of live chickens, pathological diagnosis of dead chickens, or pollution detection of the surrounding environment, this invention provides an effective detection method, offering comprehensive technical support for the prevention and control of avian infectious anemia.

[0037] V. Low cost and high efficiency

[0038] This invention optimizes the reaction system, reducing the amount of primers and probes used and eliminating the need for expensive laboratory equipment such as PCR instruments and microplate readers, thus lowering detection costs. Simultaneously, the kit components are pre-prepared, making it easy to use and reducing operational steps and reagent waste. Furthermore, the detection process is rapid and efficient, saving time and labor costs. These factors combined not only improve detection efficiency but also reduce detection costs, giving this invention a significant cost-effectiveness advantage, making it suitable for large-scale detection and widespread application. For actual production units such as chicken farms, it enables the detection and control of avian infectious anemia virus at a lower cost, helping to improve breeding efficiency and protect flock health.

[0039] VI. Good stability and reliability

[0040] The components of the kit, such as the one-step virus release solution, DNA-TERA Mix, and activator, have been optimized and validated, exhibiting excellent stability. Under specified storage conditions, the reagents have a long shelf life and stable performance. Furthermore, the detection method has been validated through multiple experiments, demonstrating good repeatability and high reliability. Stable and reliable detection results can be obtained whether testing is performed between different batches of kits or at different times and in different laboratories using the same kit. This stability and reliability provide strong assurance for the detection of chicken infectious anemia virus, enabling the kit of this invention to function stably in various practical application environments.

[0041] VII. Effectively reduce reliance on large laboratory instruments

[0042] Traditional detection methods often rely on large laboratory instruments, such as PCR machines and gel imaging systems. These instruments are not only expensive but also complex to operate and require specific laboratory conditions. This invention, through isothermal amplification and lateral flow detection technology, enables detection reactions in simple equipment such as isothermal water baths or isothermal metal baths, effectively eliminating reliance on large instruments. This innovative detection method allows detection to be conducted in various environments, including grassroots laboratories and field operations, greatly improving the flexibility and accessibility of detection and providing a more convenient technical means for the rapid diagnosis and prevention of infectious anemia in chickens.

[0043] In summary, this invention offers numerous advantages, including speed and convenience, high sensitivity and specificity, visual result interpretation, broad sample applicability, cost-effectiveness, good stability and reliability, and effective independence from large laboratory instruments. These advantages make this invention valuable for the detection and prevention of infectious anemia in chickens and give it broad market prospects, providing strong technical support for the healthy development of the poultry industry. Attached Figure Description

[0044] Figure 1 Primer and probe screening for the chicken infectious anemia virus isothermal amplification detection kit; A: negative sample; B: positive sample.

[0045] Figure 2 For the determination of primers and probes; A: 4 batches of negative samples; B: 4 batches of positive samples (10 copies); C: 4 batches of positive samples (2 copies).

[0046] Figure 3 To determine the optimal primer and probe amounts; A: First experiment; B: Second experiment; The 1, 2, 3, 4, and 5 marked above (first row) represent the combinations of 5 primer and probe amounts (see Table 3.4), and the 10 and 2 marked below (second row) represent 10 copies and 2 copies, respectively. NTC represents the negative control.

[0047] Figure 4 The results represent the optimal reaction temperature.

[0048] Figure 5 The results are from studies on optimal reaction time and sensitivity.

[0049] Figure 6 Results of specificity studies for the chicken infectious anemia virus isothermal amplification detection kit: 1. ILTV; 2. FAdV-4; 3. FAdV-8a; 4. FAdV-8b; 5. FAdV-11; 6. MDV; 7. IBV; 8. H3 subtype AIV; 9. H6 subtype AIV; 10. H9 subtype AIV; 11. NDV; 12. IBDV. Detailed Implementation

[0050] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0051] Example

[0052] 1. Preparation of positive control (PTC) and negative control (NTC) samples for chicken infectious anemia virus

[0053] 1.1 Inoculation of positive and negative samples: A strain 2823 of chicken infectious anemia virus (provided by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences) was diluted 100 times with sterile PBS and inoculated into the yolk sacs of 15 5-day-old SPF chicken embryos as the positive group. At the same time, an uninoculated chicken embryo was set up as the negative group. The positive and negative chicken embryos were placed in an incubator at 37°C for 14 days and candled every 24 hours. Chicken embryos that died within 14 days after inoculation were discarded.

[0054] 1.2 Harvesting of Positive and Negative Samples On days 5, 10, and 14 post-inoculation, five chicken embryos from each group were aseptically removed using sterile forceps in a biosafety cabinet, transferred to sterile grinding tubes, and weighed. Sterile PBS (1 mL PBS per gram of embryo) was added to the grinding tubes, and the tubes were ground in a tissue homogenizer for 90 seconds at a frequency of 180 Hz. After the embryo and PBS mixture was homogenized, the grinding tubes were placed in a centrifuge at 3000 r / min for 10 min, and the supernatant was harvested. Samples were taken for nucleic acid content determination, and the optimal harvest time was determined based on the viral nucleic acid content at different times.

[0055] 1.3 Determination of Nucleic Acid Content of Chicken Infectious Anemia Virus Take 10 μL of virus solution and add it to a centrifuge tube containing 90 μL of one-step virus release solution (Sangon Biotech (Shanghai) Co., Ltd., catalog number: B518412) (sample: release solution 1:9). Incubate at room temperature for 5-15 minutes. Take an appropriate amount of the extracted product according to the amplification reagent system and directly perform nucleic acid amplification. Detect the extracted viral nucleic acid content using the qPCR method established in this invention. Prepare the reaction system in an 8-tube real-time PCR instrument (Table 1.1). Place the reaction tube in an ABI 7500 real-time PCR instrument, set the reaction program (Table 1.2), and perform the detection. After the reaction is complete, calculate the viral nucleic acid content.

[0056]

[0057] The 2×Super Abstart Probe qPCR Mix was sourced from Sangon Biotech (Shanghai) Co., Ltd., catalog number: B630015-0001, and its universal primer sequences are as follows:

[0058] CIAV qPCR F: 5'-CTCGAAGAAGCGATCCTGCG-3' (SEQ ID NO: 12);

[0059] CIAV qPCR R: 5'-TGCCASCGTCCTCTTCTG-3' (SEQ ID NO: 13);

[0060] CIAV qPCR P: 5'-FAM-CCACTRCTCCCAGCCGACCCCG-BHQ1-3' (SEQ ID NO: 14).

[0061]

[0062] 1.4 Results A strain 2823 of chicken infectious anemia virus (TIV) was diluted 100-fold and inoculated into the yolk sacs of 5-day-old SPF chicken embryos as the positive group. A PBS inoculation group was established as the negative group. On days 5, 10, and 14 post-inoculation, chicken embryos were collected, ground, centrifuged, and nucleic acid was extracted for qPCR detection. The results showed that the TAV nucleic acid content in the homogenate of the positive group chicken embryos on days 5, 10, and 14 post-inoculation was 10... 3.7 Copy / 0.1 g, 10 4.6 Copy / 0.1 g and 10 8.1 The negative group samples were all negative (Table 1.3), thus determining that chicken infectious anemia virus should be harvested on the 14th day after inoculation of 5-day-old chicken embryos into the yolk sac, and a batch of positive and negative samples should be prepared according to this condition.

[0063]

[0064] 2. Virus inactivation and testing

[0065] 2.1 Virus Inactivation: Formaldehyde solution was added to the harvested virus solution to a final formaldehyde concentration of 0.2%. The virus-formaldehyde mixture was inactivated at 36-37°C for 24 hours, with shaking every 8 hours. Six 5-day-old SPF chicken embryos were inoculated with 0.2 mL of the inactivated virus solution and incubated at 37°C. Samples were collected at the harvest time determined by method 1.2. After mixing the samples, one blind passage was performed in the chicken embryos using the same method. Non-specifically dead chicken embryos within 24 hours were discarded from both generations of inoculated embryos, and the nucleic acid content of chicken infectious anemia virus in each generation sample was detected.

[0066] 2.2 Results: The prepared positive samples were inactivated using formaldehyde at a final concentration of 0.2%. qPCR analysis showed that the nucleic acid content of the inactivated positive samples was 10... 7.5 0.1 g (Table 2.1). The inactivated samples were inoculated into 5-day-old SPF chicken embryos and passaged blindly for 1 generation. The nucleic acid content of chicken infectious anemia virus in the ground embryo samples of the two generations of chicken embryos was detected by qPCR. No virus was detected in either sample (Table 2.1), indicating that the virus was successfully inactivated in the positive samples.

[0067]

[0068] Note: "-" indicates a negative result.

[0069] 3. Determination of primers and probes

[0070] 3.1 Primer and Probe Design and Synthesis The nucleic acid sequences of 2823 strains of chicken infectious anemia virus (CIV) and 250 other CIV strains indexed in NCBI were compared and analyzed using SnapGene software. Nucleotide fragments with good conservation were selected as target regions for isothermal amplification reactions. After determining suitable target regions, four upstream primers (F1, F2, F3, and F4), three probes (P1, P2, and P3), and four downstream primers (R1, R2, R3, and R4) (Table 3.1) were designed sequentially using SnapGene software as candidate primers and probes, and synthesized by Sangon Biotech (Shanghai) Co., Ltd. All primers and probes were purified by HPLC.

[0071]

[0072] 3.2 Screening of Optimal Primers After synthesizing candidate primers, different combinations of candidate upstream primers, probes, and downstream primers were used (Table 3.2) to detect chicken infectious anemia virus (CIV) negative and positive samples using isothermal amplification. First, primer combinations with high specificity for detecting negative samples were screened. Further screening was conducted on primer combinations that produced darker detection bands for positive samples. Through this two-step screening, the performance of different candidate primer combinations was determined, thereby identifying the optimal primer and probe combinations for further experiments.

[0073]

[0074] 3.3 Isothermal Amplification Detection Method for Chicken Infectious Anemia Virus First, the recombinase polymerase amplification (RPA) reaction was performed according to the instructions of the chromatographic DNA isothermal amplification kit (TERA method) (Sangon Biotech (Shanghai) Co., Ltd., catalog number: Sangon B631002). The specific operation is as follows:

[0075] 1) Prepare the reaction reagents according to the number of samples to be tested. Take the reaction reagents out from -20℃ and allow them to equilibrate at room temperature for 10 min.

[0076] 2) Prepare the RPA reaction mixture in an enzyme-free centrifuge tube according to the required volumes of upstream primer, downstream primer, probe, template, activator and ddH2O for each reaction (Table 3.3).

[0077] 3) After preparation, immediately add 25 μL of the prepared reaction mixture to each reagent reaction tube, and immediately and quickly invert the reaction tube up and down 8 to 10 times or gently shake to mix.

[0078] 4) After mixing, shake (or centrifuge quickly) the reaction solution to the bottom of the reaction tube, and then immediately place the reaction tube into a constant temperature device (in this example, the Dongshenglong 821 PCR instrument is used).

[0079] 5) Set the reaction program to 40℃ and the reaction time to 15 min.

[0080] 6) After the reaction is complete, add 5 μL of the reaction solution to a centrifuge tube containing 95 μL of ddH2O, mix well, and then drop 80 μL of the dilution solution into the sample port of the nucleic acid chromatography test strip for chromatography detection.

[0081] 7) After standing for 10-15 minutes, observe the control line (C line) and the test line (T line) and interpret the results.

[0082] 8) Judgment criteria;

[0083] Negative: Test strip C shows one red band, and T shows no band; Positive: Test strip C shows one red band, and T shows one red band. The T band can be either dark or light, all of which indicate a positive result; Invalid: Test strip C does not show a band, and regardless of whether T shows a band, the result is invalid and must be tested again.

[0084] 9) After recording the test results, seal the test strip and discard it in a safe place.

[0085]

[0086] 3.4 Determination of the optimal primer and probe combination After screening multiple sets of candidate primer combinations with high specificity and good reactivity, it is necessary to further determine the candidate primer combination based on the conservation of the sequence, and repeat the determined primer combination multiple times to ensure that the reactivity, specificity and reproducibility of the primer and probe are good.

[0087] 3.5 Determination of Primer and Probe Dosage To minimize detection costs, based on the determined upstream, downstream, and probe combinations used in the RPA reaction, the dosage of primers and probes in the reaction system was further optimized (Table 3.4). Under different primer and probe dosage conditions, the reaction temperature was set to 40 ℃ and the reaction time to 15 min. Two templates with low nucleic acid content (10 copies and 2 copies) were detected using the detection method. The optimal primer and probe dosages were determined based on the reaction results.

[0088]

[0089]

[0090] 3.6 Screening of Optimal Primers Twenty-eight combinations of the synthesized four upstream primers, three probes, and four downstream primers (Table 3.2) were used for the isothermal amplification detection method for chicken infectious anemia virus (CIV). First, CIV-negative samples were tested using the CIV isothermal amplification detection method. It was found that all 28 primer and probe combinations produced negative results (Figure 1A), indicating that these 28 primer and probe combinations did not produce false positives and had good specificity. When these 28 primer and probe combinations were used to detect positive samples, all samples tested positive (Figure 1B) when combinations 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 21, 23, 25, 26, 27, and 28 were used (Figure 1B). Further comparison of the color development of the test line (T line) in these positive reaction results revealed that the T line bands of reaction groups 4, 5, 6, 26, and 28 were darker. Figure 1 B), which is more suitable as a candidate combination. Finally, we compared the sequences of these 5 combinations with the sequences of multiple CIAV strains. According to the sequence alignment results, we found that the primers and probes of the fourth group had higher conservation for different chicken infectious anemia virus nucleotide sequences. Therefore, the primers and probes of the fourth combination were selected for subsequent experiments.

[0091] To further determine the stability of the primers and probes in the fourth combination, this application used the fourth combination of primers and probes to retest four batches of negative samples, and the results were all negative. Figure 2 A). Furthermore, using the fourth primer and probe combination, four batches of positive samples with different nucleic acid contents (10 copies and 2 copies) were retested, and all results were positive. Figure 2(B, 2C). These results further demonstrate that the combination of primers and probes in group 4 has good reproducibility and high sensitivity. Based on these results, we have established the method for isothermal amplification detection of chicken infectious anemia virus using primers and probes in this application. The specific information of the primers and probes is as follows:

[0092] The nucleotide sequence of the upstream primer is as follows:

[0093] 5'-TGGTATCGCTGGAATTACAATCRCTCTATC-3', R=A / G, as shown in SEQ ID NO:1;

[0094] The nucleotide sequence of the downstream primer is as follows:

[0095] 5'-TTCTTCGAGGGAGGCTTGGSTTGATCGGTC-3', S=C / G, as shown in SEQ ID NO:8, the 5' end is modified with biotin;

[0096] The nucleotide sequence of the probe is as follows:

[0097] 5'-CGAATGCTCGCGCTCCCACGCTAAGATCTG[THF]AACTGCGGACAATTC-3', as shown in SEQ ID NO:6, has FAM modified at the 5' end, C3Spacer modified at the 3' end, and tetrahydrofuran (THF) modified at the 31bp midpoint of the 5' end.

[0098] The 5' and 3' ends of the downstream primers and probes for RPA contain different modifications, and the purification of the primers and probes using HPLC results in high synthesis costs. To reduce subsequent detection costs, this application further optimized the amount of primers and probes used in the reaction system. Under different primer and probe usage conditions, the RPA-LFD method was used to detect two templates with different nucleic acid contents (10 copies and 2 copies), with each condition repeated twice. Figure 3 (A, 3B). The results showed that all five combinations could detect 10-copy and 2-copy nucleic acid samples. Among them, group 5 (1.4 μL upstream primer, 1.4 μL downstream primer, 0.42 μL probe) used the largest amounts of primers and probe, but had the worst detection effect; group 1 (0.6 μL upstream primer, 0.6 μL downstream primer, 0.18 μL probe) used the smallest amounts of primers and probe, but had the best detection effect.

[0099] 4. Determination of the optimal reaction temperature

[0100] 4.1 Method: Prepare lyophilized pellets A and B from the aforementioned reagents (Table 4.1). Reset the dropper, rinse the dropper twice with 75% alcohol and water respectively, drain the liquid from the tube, and insert the inlet tube into the reagent tube to be dispensed. Inject the liquid, replacing the dropper with 15.5 μL and 6 μL dropper heads respectively, depending on the reagent volume. Perform an accuracy test (dispose of the first two drops as waste liquid), until the mass is equal to the reagent reaction volume. Pour liquid nitrogen into a thermos and place it on the lyophilization equipment, dispensing the pellets in automatic mode. After dispensing, pour the pellets into a lyophilization tray and place it in the lyophilizer. Start lyophilization; the lyophilization program is shown in Table 4.2. After lyophilization, in a vacuum glove box, sequentially load lyophilized pellets A and B into the reaction tubes, cover the reaction tubes, and place them in an aluminum foil bag for vacuum packaging.

[0101] To determine the optimal reaction temperature for the dried reaction reagents, negative samples of chicken infectious anemia virus (CIV) and positive samples with different nucleic acid contents (100 copies, 10 copies, and 2 copies) were added to the dried reaction tubes. The prepared reaction systems were then subjected to isothermal amplification reactions at 38℃, 40℃, 42℃, and 44℃, with a uniform reaction time of 15 min. Based on the specificity and sensitivity of detecting CIV nucleic acid under different reaction temperature conditions, the optimal reaction temperature was determined.

[0102]

[0103]

[0104] 4.2 Results To determine the optimal reaction temperature after low-temperature lyophilization of the reagents, this application added chicken infectious anemia virus (RIV) negative samples and positive samples with different nucleic acid contents (100 copies, 10 copies, and 2 copies) to the dried reaction tubes. Subsequently, the prepared reaction system was subjected to RPA reactions at 38℃, 40℃, 42℃, and 44℃, with a uniform reaction time of 15 min. The results showed that no false positive reactions occurred with the negative samples at any of the four reaction temperatures, further proving that low-temperature lyophilization does not lead to false positives. For the 100-copy positive sample, there was no significant difference in detection results at all four temperatures. Figure 4 However, while 10 copies of a positive sample could be detected at 38°C and 40°C, no visible color reaction was observed in the test line when detecting 2 copies of a positive sample, and therefore the result was judged as negative. Figure 4 In contrast, at 42℃ and 44℃, both 10-copy and 2-copy positive samples showed a visible colorimetric reaction at the T line. Figure 4The result was determined to be positive. Taking into account both the sensitivity of the test and its practicality for on-site testing, this application ultimately determined 42℃ as the optimal reaction temperature for the isothermal amplification detection method of chicken infectious anemia virus. This temperature not only ensures the accuracy of the test results but also improves the sensitivity of the test, making it suitable for rapid on-site detection.

[0105] 5. Determination of optimal reaction time and sensitivity study

[0106] 5.1 Methods To further determine the optimal reaction time under optimal reaction temperature conditions, avian infectious anemia virus (AID) negative samples and positive samples with different nucleic acid contents (100 copies, 10 copies, and 2 copies) were added to the dried reaction tubes. The prepared reaction systems were then subjected to isothermal amplification reactions at the optimal reaction temperature for 10 min, 12 min, 15 min, and 20 min, respectively. Based on the sensitivity of the isothermal amplification detection method for AID nucleic acid under different reaction time conditions, the optimal reaction time was determined.

[0107] 5.2 Results To further determine the optimal reaction time and sensitivity of the dried reaction reagent at 42℃, this application added chicken infectious anemia virus (RIV) negative samples and positive samples with different nucleic acid contents (100 copies, 10 copies, and 2 copies) to the dried reaction tubes. Subsequently, the prepared reaction system was placed at 42℃ for RPA reaction, with four different reaction times set: 10 min, 12 min, 15 min, and 20 min. The results showed that no false positives occurred in the negative samples at reaction times of 10 min, 12 min, and 15 min. Figure 5 However, at a reaction time of 20 minutes, negative samples showed false positives. Figure 5 The results of testing positive samples are as follows: At 10 min of reaction time, no positive result was detected in 100 copies of nucleic acid sample. Figure 5 At 12 min, both 100-copy and 10-copy nucleic acid samples were judged to be positive, but the T-line colorimetric reaction was weak. Figure 5 At a reaction time of 15 min, both 100-copy and 10-copy nucleic acid samples were deemed positive, and the T-line was clearly visible. These results indicate that the sensitivity of the detection increases with reaction time, but excessively long reaction times can lead to false positives. To ensure the accuracy and sensitivity of on-site testing, this application ultimately determined the reaction time to be 15 min and the limit of detection to be 10 copies.

[0108] 6. Specificity studies

[0109] 6.1 Methods 2823 strains of chicken infectious anemia virus (PTC) were used as positive controls, and SPF chicken tissue (mixed homogenate of heart, liver, spleen, lung, kidney, bursa of Fabricius, and thymus) was used as negative controls (NTC). Twelve other common chicken-derived viruses were used as specific quality control samples, including infectious laryngotracheitis virus (ILTV), avian adenovirus type 4 (FAdV-4), avian adenovirus type 8a (FAdV-8a), avian adenovirus type 8b (FAdV-8b), avian adenovirus type 11 (FAdV-11), Marek's disease virus (MDV), infectious bronchitis virus (IBV), H3 subtype avian influenza virus (H3 subtype AIV), H6 subtype avian influenza virus (H6 subtype AIV), H9 subtype avian influenza virus (H9 subtype AIV), Newcastle disease virus (NDV), and infectious bursa of Fabricius virus (IBDV). Three batches of laboratory-developed kits were used to test the specific quality control samples, and the experimental results were recorded and analyzed.

[0110] 6.2 Results The specific quality control samples were tested using three batches of a rapid nucleic acid detection kit for chicken infectious anemia virus (TIV), which was developed in the laboratory. The results showed that the positive control for TAV was positive, the negative control (SPF chicken tissue) was negative, and all other virus samples were negative. Figure 6 This indicates that the kit has good specificity.

[0111] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A primer and probe for rapid detection of chicken infectious anemia virus by isothermal amplification, characterized in that, the nucleotide sequence of the upstream primer is: 5'-TGGTATCGCTGGAATTACAATCRCTCTATC-3', R=A / G, as shown in SEQ ID NO: 1; the nucleotide sequence of the downstream primer is: 5'-TTCTTCGAGGGAGGCTTGGSTTGATCGGTC-3', S=C / G, as shown in SEQ ID NO: 8, with Biotin modification at the 5' end; the nucleotide sequence of the probe is: 5'-CGAATGCTCGCGCTCCCACGCTAAGATCTG[THF]AACTGCGGACAATTC-3', as shown in SEQ ID NO: 6, with FAM modification at the 5' end, C3Spacer modification at the 3' end, and tetrahydrofuran THF modification at the 31bp position away from the 5' end.

2. The Infectious Bursal Disease Virus constant temperature amplification rapid detection kit, characterized in that, The primer and probe of claim 1.

3. The kit of claim 2, wherein The kit further comprises an activator, a nucleic acid chromatography test strip, a positive control sample, and a negative control sample.

4. The kit of claim 3, wherein The activator is 21mM magnesium acetate; the positive control sample is chicken infectious anemia virus nucleic acid, and the negative control sample is SPF chicken embryo grinding liquid nucleic acid.

5. The primer and probe of claim 1 or the kit of any one of claims 2-4 for use in detecting chicken infectious anemia virus.

6. Use according to claim 5, characterized in that, The samples for detection include live chicken samples, necropsy samples of dead chickens, slaughterhouse necropsy samples, and samples of the surrounding environment contaminated by sick chickens; The temperature for isothermal amplification is 42°C, and the time is 15 minutes.

7. A method for rapid detection of chicken infectious anemia virus by isothermal amplification, characterized by, The detection is achieved by the primer and probe of claim 1 or the kit of any one of claims 2-4, comprising: mixing the sample nucleic acid with a reaction solution containing the primer and probe, then immediately placing the reaction tube into a 42°C constant temperature device for 15 minutes, after the reaction is completed, diluting the reaction solution with ddH2O, then dropping the diluted solution into the sample port of the nucleic acid chromatography test strip for chromatographic detection, and observing the quality control line C and the detection line T after standing for 10-15 minutes to determine the result.

8. The method of claim 7, wherein, If a band appears on the test strip C line and no band appears on the negative control sample T line, and a red band appears on the positive control sample T line, the test result is valid; If no band appears on the test strip C line, regardless of whether a band appears on the T line, the result is invalid and needs to be retested; Under the condition that the test is valid, if a red band appears on the test strip C line added with the sample to be tested and no band appears on the T line, the sample is determined to be negative for chicken infectious anemia virus nucleic acid; if a red band appears on the test strip C line added with the sample to be tested and a red band appears on the T line, regardless of the color depth, the sample is determined to be positive for chicken infectious anemia virus nucleic acid.

9. The method of claim 7, wherein, The reaction is carried out according to the following reaction system: 。 10. The method of claim 7, wherein, The method for obtaining sample nucleic acid comprises: i. Live chicken sample: 5mL of anticoagulated blood or serum is collected from a live chicken under sterile conditions, and 200 μL of the sample is taken for nucleic acid extraction; ii. Necropsy sample of a dead chicken or slaughterhouse necropsy sample: a tissue sample of a dead chicken is collected under sterile conditions, the sample to be tested is cut with sterile scissors and forceps into a grinding tube, and nucleic acid extraction is performed; iii. Peripheral environment samples contaminated by sick chickens: Take the feces of the place related to sick chickens into the one-step virus release solution to make 10% homogenate solution, and perform nucleic acid extraction; or directly take 200 μL sewage to perform nucleic acid extraction.

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