A super multiplex digital PCR detection method for simultaneous detection of multiple pathogens in shrimp and its application

Abstract

This application relates to a supermultiplex digital PCR detection method and its application for the simultaneous detection of multiple pathogens in shrimp. The digital PCR primers, probes, kits, and detection method for shrimp pathogen detection in this application can rapidly, accurately, and ultrasensitively detect pathogens affecting 18 types of shrimp. By detecting pathogens in shrimp, the health status of shrimp in intensive shrimp farming can be assessed in a timely manner, disease transmission can be prevented, and timely intervention measures can be taken to reduce the risk of disease transmission to shrimp populations in intensive farming, avoiding serious economic losses. This method has extremely high application value. Accurate identification of specific pathogens in shrimp samples is crucial for designing appropriate control strategies, as each pathogen may require different prevention and management methods, effectively reducing the need for drug use. Shrimp pathogen detection can improve farming efficiency and also contribute to the prevention and control of aquatic animal diseases and the inspection and quarantine monitoring programs for seedlings of major aquaculture species.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to primers, probes, methods, and kits for the simultaneous detection of multiple pathogens in shrimp using ultramultiplex digital PCR. Background Technology

[0002] Shrimp pathogens include bacteria, viruses, parasites, and protozoa. Viruses are the most important pathogenic factors in shrimp, and are divided into DNA viruses and RNA viruses. DNA viruses mainly include ① White Spot Syndrome Virus (WSSV), which causes white spot disease (WSD). WSSV is a double-stranded DNA virus and is considered one of the most serious of the approximately 20 viral pathogens affecting shrimp. It is also one of the main viral pathogens causing harm to farmed shrimp in my country and the Asia-Pacific region in recent years. It can infect the vast majority of shrimp species with a high mortality rate. In addition, it can also infect various crustaceans such as crabs, lobsters, amphipods, and water flies in freshwater and marine ecosystems, exhibiting a wide host range and causing serious economic losses to the aquaculture industry. ② Another DNA virus is Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), which is the smallest known shrimp virus and an important viral pathogen affecting shrimp, causing severe economic losses to the shrimp farming industry (up to 50% of the overall economic losses in shrimp farming). IHHNV is a single-stranded DNA virus. IHHNV causes poor growth, irregular growth, and epidermal deformities in shrimp at harvest. The resulting disease is infectious subcutaneous and hematopoietic necrosis, listed as an important crustacean disease requiring reporting by the World Organisation for Animal Health (OIE). ③ Penaeus monodon baculovirus (MBV). ④ Shrimp hemocytosis virus (SHIV) is a newly discovered iridovirus that infects aquatic crustaceans. SHIV can be transmitted horizontally through feces and cannibalism. Infected Litopenaeus vannamei exhibit clinical symptoms such as anorexia, empty gastrointestinal tract, pale hepatopancreas, and death. SHIV is widespread in major shrimp farming areas in China, infecting not only white shrimp but also giant freshwater prawns, Penaeus monodon, Japanese shrimp, and freshwater prawns. ⑤ Shrimp hepatopancreas parvovirus (HPV): Infected larvae become lethargic, have reduced appetite, grow slowly, molt infrequently, and often have dirt or symbiotic organisms attached to their bodies. During the farming period, juvenile or adult shrimp are thin, dark in color, and have numerous black spots on their carapace. Some have softened carapaces and whitened abdominal muscles. They have poor resistance and are prone to secondary bacterial infections.

[0003] RNA viruses include: ① Infectious Myonecrosis Virus (IMNV), which causes Infectious Myonecrosis (IMN), is a double-stranded RNA virus. Typical symptoms of IMNV infection include decreased transparency, necrosis of the abdomen and cephalothorax, tail discoloration, shrinkage of the hepatopancreas, and gradual necrosis of the tail fan. Since there are no effective drugs or vaccines for IMNV, sensitive and reliable diagnosis is necessary for appropriate control measures; therefore, prevention, management, and timely diagnosis are the most effective means. ② Yellow Head Disease Virus (YHV), which causes Yellow Head Disease (YHD), is a single-stranded RNA virus. Its envelope glycoprotein (gp116) has been shown to be the main virulence factor of YHV genotype 1. Acute infection in shrimp manifests as loss of appetite within 2-4 days, with a high mortality rate. Dying shrimp develop a yellow cephalothorax due to yellowing of the hepatopancreas, hence the name Yellow Head Disease. In my country, it is classified as a Category II disease, while the OIE lists it as a disease requiring mandatory reporting. ③ Taura Syndrome Virus (TSV) is considered one of the three most serious shrimp viruses, alongside WSSV and YHV. TSV is a single-stranded RNA virus. ④ MrNV, the nodavirus that causes white tail disease (WTD) in giant freshwater prawns, is a single-stranded RNA virus composed of two segments: RNA-1 and RNA-2. MrNV can infect through both horizontal and vertical transmission. Vertical transmission from infected broodstock prawns is the main cause of disease in larvae; it can also be transmitted horizontally through contaminated water, rotifers and other biological feed, tools, and inadequately disinfected larval ponds. ⑤ XSV, a satellite virus related to MrNV, is a single-stranded positive RNA virus first discovered in Thailand and prevalent in several countries and regions. Co-infection with XSV and MrNV can lead to mass mortality of giant freshwater prawn larvae within a short period, and the muscles of infected prawns exhibit a whitish tinge. ⑥ CMD, caused by Nodamura virus (CMNV), is a single-stranded RNA virus. Infected prawns mainly die successively in the deep water at the bottom of the pond, making it difficult to observe, hence the name CMD or bottom death disease. Diseased individuals exhibit symptoms such as a lighter-colored and atrophied hepatopancreas, empty intestines and stomachs, and slow growth. Often, the abdominal muscles of diseased shrimp are also opaque or locally whitish. Mortality rates increase in diseased shrimp at higher water temperatures (above 28℃), with cumulative mortality reaching up to 80%.

[0004] Pathogenic bacteria include the virulence factor PirA / B, which causes acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by a sudden, large-scale mortality (up to 100%) within a short period (usually within 30-35 days after the introduction of juvenile or young shrimp into the rearing tank). Larger juvenile shrimp may also be affected. In areas where AHPND is endemic in shrimp farms, evidence suggests an infection rate approaching 100%. Disease signs in dying shrimp include a pale to white coloration of the hepatopancreas due to loss of pigment in the connective tissue, and behavioral changes such as frequent slumping to the bottom may also be observed. Other pathogens include Vibrio parahaemolyticus and Vibrio harveyi, Vibrio phycoides causing vibriosis, Streptococcus ginseng causing streptococcal disease, and Pseudomonas fluorescens causing infection and death.

[0005] Enterocytozoon hepatisimidae (EHP) is an obligate intracellular parasite. EHP infection causes slow growth in shrimp and severe economic losses. It is a new and important pathogen that has been threatening the development of the shrimp industry in recent years.

[0006] Detection of shrimp pathogens often requires speed, efficiency, comprehensiveness, and accuracy, but current nucleic acid detection methods cannot meet these requirements. Various molecular detection methods also have significant limitations. National and industry standards for pathogen detection mostly employ PCR, nested PCR, or quantitative real-time PCR. However, PCR has a low limit of detection, nested PCR is cumbersome, has limited detection capabilities, and is prone to nucleic acid contamination. Quantitative real-time PCR uses dyes to detect pathogens, making quality control of the extraction process impossible, and the use of melting curve analysis when multiple indicators are involved can easily lead to misinterpretations. Quantitative real-time PCR using the TagMan probe method requires a standard curve for quantification and demands high primer and probe amplification efficiency; most four-channel quantitative real-time PCR instruments cannot detect more than four pathogens per tube.

[0007] Establishing a rapid, efficient, comprehensive, accurate, highly sensitive, and highly specific method for detecting multiplex resistant pathogens is crucial not only for the diagnosis and prevention of shrimp diseases but also for guiding treatment and safe medication management. Efficient and rapid detection of pathogenic microorganisms in farmed shrimp can improve farming efficiency and reduce economic losses, demonstrating significant application value. Summary of the Invention

[0008] The purpose of this application is to provide ultra-multiplex PCR primer pairs, probes, microfluidic cartridges, kits, detection systems, and detection methods for the specific detection of shrimp pathogens and parasites. These methods enable rapid, accurate, and ultra-high sensitivity detection of 18 pathogens and virulence factors causing diseases in farmed shrimp. The PCR primer pairs, probes, microfluidic cartridges, kits, detection systems, and detection methods can be used in any PCR detection system, including but not limited to conventional PCR, RL-PCR, RNA-PCR, quantitative real-time PCR, digital PCR, PCR-ELISA, nested PCR-high resolution melting analysis (nPCR-HRM), etc., with digital PCR being preferred. For example, the purpose of this application is to provide PCR primer pairs, probes, microfluidic cartridges, kits, detection systems, and detection methods for the specific detection of sequences.

[0009] To achieve the above objectives, this application adopts the following technical solution:

[0010] This application relates to a combination for the specific detection of target genes, wherein the combination includes 18 pairs of primers for amplifying 18 target genes and 18 probes for hybridizing the 18 genes. Five probes are labeled with three concentrations of two different fluorescent groups, and each of the five probes carries a different type or concentration of fluorescent group. Simultaneously, one probe is labeled with one concentration of one fluorescent group, so as to realize the simultaneous detection of 11 target genes in one well of the chip using five fluorescent groups.

[0011] In one specific implementation, the 18 primers and probes include any one or both of the following two sets of primers and probes:

[0012] The first set of primers and probes comprises: a PCR primer and probe combination for specifically amplifying a specific fragment of the virulence factor PirA / B, wherein the primers include the upstream primer shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:2, and the probe includes the probe shown in SEQ ID NO:3; a PCR primer and probe combination for specifically amplifying a specific fragment of WSSV virus, wherein the primers include the upstream primer shown in SEQ ID NO:4 and the downstream primer shown in SEQ ID NO:5, and the probe includes the probe shown in SEQ ID NO:6; a PCR primer and probe combination for specifically amplifying a specific fragment of IMNV virus, wherein the primers include the upstream primer shown in SEQ ID NO:7 and the downstream primer shown in SEQ ID NO:8, and the probe includes the probe shown in SEQ ID NO:9; and a PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio parahaemolyticus VP, wherein the primers include the upstream primer shown in SEQ ID NO:10 and the downstream primer shown in SEQ ID NO:11, and the probe includes the probe shown in SEQ ID NO:11. The probe shown in NO:12; a PCR primer and probe combination for specifically amplifying a specific fragment of TSV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:13 and a downstream primer as shown in SEQ ID NO:14, and the probe includes the probe shown in SEQ ID NO:15; a PCR primer and probe combination for specifically amplifying a specific fragment of IHHNV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:16 and a downstream primer as shown in SEQ ID NO:17, and the probe includes the probe shown in SEQ ID NO:18; a PCR primer and probe combination for specifically amplifying a specific fragment of shrimp enterocytozoon hepaticus EHP, wherein the primers include an upstream primer as shown in SEQ ID NO:19 and a downstream primer as shown in SEQ ID NO:20, and the probe includes the probe shown in SEQ ID NO:21; a PCR primer and probe combination for specifically amplifying a specific fragment of the internal reference gene GAPDH, wherein the primers include an upstream primer as shown in SEQ ID NO:22 and a downstream primer as shown in SEQ ID NO:23, and the probe includes the probe shown in SEQ ID NO:24.

[0013] The second set of primers and probes comprises: a PCR primer and probe combination for specifically amplifying a specific fragment of YHV virus, wherein the primers include the upstream primer shown in SEQ ID NO:25 and the downstream primer shown in SEQ ID NO:26, and the probe includes the probe shown in SEQ ID NO:27; a PCR primer and probe combination for specifically amplifying a specific fragment of MBV virus, wherein the primers include the upstream primer shown in SEQ ID NO:28 and the downstream primer shown in SEQ ID NO:29, and the probe includes the probe shown in SEQ ID NO:30; a PCR primer and probe combination for specifically amplifying a specific fragment of SHIV virus, wherein the primers include the upstream primer shown in SEQ ID NO:31 and the downstream primer shown in SEQ ID NO:32, and the probe includes the probe shown in SEQ ID NO:33; and a PCR primer and probe combination for specifically amplifying a specific fragment of HPV virus, wherein the primers include the upstream primer shown in SEQ ID NO:34 and the downstream primer shown in SEQ ID NO:35, and the probe includes the probe shown in SEQ ID NO:27. The probe shown in SEQ ID NO:36; a PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio harveyi VH, wherein the primers include the upstream primer shown in SEQ ID NO:37 and the downstream primer shown in SEQ ID NO:38, and the probe includes the probe shown in SEQ ID NO:39; a PCR primer and probe combination for specifically amplifying a specific fragment of MrNV virus, wherein the primers include the upstream primer shown in SEQ ID NO:40 and the downstream primer shown in SEQ ID NO:41, and the probe includes the probe shown in SEQ ID NO:42; a PCR primer and probe combination for specifically amplifying a specific fragment of XSV virus, wherein the primers include the upstream primer shown in SEQ ID NO:43 and the downstream primer shown in SEQ ID NO:44, and the probe includes the probe shown in SEQ ID NO:45; a PCR primer and probe combination for specifically amplifying a specific fragment of CMNV virus, wherein the primers include the upstream primer shown in SEQ ID NO:46 and the downstream primer shown in SEQ ID NO:47, and the probe includes the probe shown in SEQ ID NO:49. The probe shown in NO:48; a PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio alginate VA, wherein the primers include an upstream primer as shown in SEQ ID NO:49 and a downstream primer as shown in SEQ ID NO:50, and the probe includes a probe as shown in SEQ ID NO:51;A PCR primer and probe combination for specifically amplifying a specific fragment of Streptococcus indicus SI, wherein the primers comprise an upstream primer as shown in SEQ ID NO:52 and a downstream primer as shown in SEQ ID NO:53, and the probe comprises the probe as shown in SEQ ID NO:54; and a PCR primer and probe combination for specifically amplifying a specific fragment of Pseudomonas fluorescens PF, wherein the primers comprise an upstream primer as shown in SEQ ID NO:55 and a downstream primer as shown in SEQ ID NO:56, and the probe comprises the probe as shown in SEQ ID NO:57.

[0014] In one specific approach, the target gene is a specific fragment of a pathogenic microorganism or parasite that causes various diseases in shrimp.

[0015] In one specific manner, the pathogen fragment is selected from one or more microbial pathogens or parasite fragments selected from the following: AHPND virulence gene PirA / B, WSSV virus, IMNV virus, Vibrio parahaemolyticus VP, TSV virus, IHHNV virus, EHP, YHV virus, MBV virus, SHIV virus, HPV virus, Vibrio harveyi VH, MrNV virus, XSV virus, CMNV virus, Vibrio leucovorum VA, Streptococcus indicus SI, and Pseudomonas fluorescens PF.

[0016] In one specific approach, the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end of the probe sequence is labeled with a fluorescent quencher group; when the probes are located in the same reaction system, the spectral ranges of different types of fluorescent reporter groups are screened.

[0017] In one particular embodiment, the fluorescent group is selected from FAM, HEX, ROX, Cy5, Cy5.5, JOE, TET, TAMRA, BHQ1, BHQ2, BHQ3, and / or Eclipse.

[0018] The combination described in this application for the specific detection of 18 pathogen or virulence factor sequences is a combination used in digital PCR (dPCR) reactions.

[0019] This application also relates to a digital PCR reaction premix for the specific detection of pathogen or virulence factor sequences, wherein the digital PCR reaction premix contains the combination described in this application for the specific detection of 18 pathogen or virulence factor sequences.

[0020] In one specific embodiment, the digital PCR reaction premix further includes four, five, six, seven, eight, or nine components selected from the following: hot-start Taq enzyme, reverse transcriptase, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, and NH4. +and Mg 2+ ion.

[0021] This application also relates to a digital PCR microfluidic cartridge, wherein the digital PCR microfluidic cartridge includes the digital PCR reaction premix for specifically detecting pathogen or virulence factor sequences as described above.

[0022] In one particular embodiment, the microfluidic cartridge includes at least one or at least two orifices.

[0023] This application also relates to a kit for detecting a target gene, wherein the kit includes the digital PCR reaction premix described above for specifically detecting pathogen or virulence factor sequences.

[0024] In one specific embodiment, the kit also includes positive control, negative control, and / or droplet-generating oil.

[0025] In one particular embodiment, the kit further includes the digital PCR microfluidic cartridge described in this application.

[0026] This application also relates to a method for detecting pathogen or virulence factor sequences for non-disease diagnosis purposes, the method comprising the following steps:

[0027] (1) Release the nucleic acid from the sample to be tested;

[0028] (2) Prepare a digital PCR amplification mixture containing the nucleic acid of the sample to be tested provided in step (1), the combination of the above-mentioned application for the specific detection of 18 pathogens or virulence factor sequences, and / or the above-mentioned application for the specific detection of pathogens or virulence factor sequences.

[0029] (3) Prepare microdroplets in a digital PCR microfluidic cartridge and perform PCR amplification reaction;

[0030] (4) Use a digital PCR microfluidic cartridge reader to read the fluorescence signal after the droplet PCR amplification reaction.

[0031] The method for detecting target genes for non-disease diagnosis described in this application refers to a sample selected from water, soil, shrimp feed, and / or shrimp tissue from a shrimp farming base.

[0032] It should be understood that, within the scope of this application, the above-described technical features and the technical features specifically described below can be combined with each other to form new or preferred technical solutions. Further details are omitted due to space limitations.

[0033] The beneficial effects of this application lie in providing a primer and probe set for detecting multiple shrimp pathogens or virulence factors. The 5' end of the probe sequences is labeled with fluorescent groups of different colors and concentrations, and the target sequences of each target gene are efficiently amplified by digital PCR, achieving absolute quantification of each target gene and directly providing the copy number per microliter (CPS / μl) of each target gene in the reaction system. Furthermore, this application also provides a method for detecting target pathogens or virulence factors for non-disease diagnosis purposes using the primer and probe set of this application. For example, the combination of this application can detect multiple pathogens or virulence factors in artificially cultured shrimp tissues. The sensitivity and specificity of this method are 95% and 100%, respectively, exceeding existing technologies; moreover, the limit of detection is 500 cps / ml, far lower than the limit of detection for direct detection of diseased shrimp tissue samples using existing qPCR techniques.

[0034] Specifically, 1) the digital PCR detection kit directly detects the nucleic acids of pathogens or virulence factors in 200 μL of shrimp tissue, eliminating the need for a separate reverse transcription step for RNA viruses, enabling simultaneous detection of DNA viruses, RNA viruses, bacteria, parasites, and virulence factors in a single step; 2) the digital PCR primer-probe set fluorescence mixing coding technology enables single-well detection of 11 pathogens; 3) the detection targets include, but are not limited to, shrimp pathogens or virulence factors; and 4) the detection kit contains an internal reference gene to prevent false negatives caused by insufficient sample extraction quality or failed PCR amplification reaction.

[0035] In one specific implementation, the types of samples involved in the testing include shrimp tissue, shrimp feed, environmental soil, and aquaculture water. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figures 1A-1F This is the positive control detection result of Example 1, which uses the primer and probe set of this application to simultaneously detect 18 pathogens and virulence factors plus one internal reference gene in 2 wells. Figure 1A , 1B 1C represents the detection results of seven pathogenic microorganisms and virulence factors detected in Well 1; Figure 1D , 1E 1F and 1F are the detection results of 11 pathogens detected in well 2.

[0038] Figures 2A-2C To optimize the detection efficiency of each shrimp pathogen by designing different primers and probes, and then select the optimal primers and probes for detection. Figure 2AThe results were obtained by amplifying PirA / B positive plasmids of the same concentration and volume using four sets of PirA / B primers and probes. Figure 2B The results are from amplification using WSSV positive plasmids of the same concentration and volume, and four sets of WSSV primers and probes. Figure 2C The results are from amplification using IMNV positive plasmids of the same concentration and volume, and four sets of IMNV primers and probes.

[0039] Figure 3 This is a specificity test result. The 18 pathogen and virulence factor detection kit reacted with 115 strains in two replicates; no positive droplets were produced in either well. This demonstrates that the 18 pathogen detection kit did not exhibit nonspecific cross-reactivity with the 115 strains.

[0040] Figures 4A-4C To use the kit of this application to detect the results of shrimp tissue samples carrying pathogenic viruses or bacteria. Figure 4A This indicates that WSSV-specific fragments were detected in shrimp carrying the WSSV virus, with the orange-marked droplets being WSSV-positive droplets; Figure 4B This indicates that a VP-specific fragment was detected in a shrimp tissue carrying Vibrio parahaemolyticus VP, with the blue-marked droplets being VP-positive droplets; Figure 4C This indicates that specific fragments of PirA / B virulence factors were detected in shrimp tissues carrying PirA / B virulence factors, with the blue-marked droplets being PirA / B positive droplets.

[0041] Figure 5 To detect the results of a WSSV-positive shrimp feed sample using the kit of this application, the orange-marked droplets are WSSV-positive droplets. Detailed Implementation

[0042] The specific embodiments of this application are described in detail below, but it should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments that do not specify specific experimental conditions are generally operated under conventional conditions or as recommended by the manufacturer.

[0043] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated ingredients or components without excluding other ingredients or other components.

[0044] Digital PCR (dPCR) is a novel absolute quantitative PCR technique. It primarily involves limiting dilution of PCR reactants, followed by PCR amplification in different reaction chambers. Finally, the initial copy number or concentration of the target molecule is determined based on the Poisson distribution principle and the number and proportion of positive droplets. Because multiplex digital PCR reaction systems contain multiple primers and probes, the requirements for primer-probe combinations in the detection system are very high.

[0045] To date, there are no reports on multiplex PCR techniques for the efficient detection of multiple pathogens (DNA viruses, RNA viruses, bacteria, and parasites) and virulence factors in mixed nucleic acid samples using digital PCR.

[0046] This application relates to a combination for the specific detection of P target genes, wherein the combination includes P pairs of primers for amplifying P sequences and P probes for hybridizing the P genes, and the P probes are labeled with N concentrations of M different fluorescent groups, wherein each of the P probes carries a different type or concentration of fluorescent group; the P target genes and the fluorescent groups satisfy the following formula: 2≤P≤(M N +M)-X, where M is a positive integer greater than or equal to 2; N is an integer from 1 to 3; X here is a value used to correct the total number of targets to ensure that the P value is reasonable, and is used to limit the actual number of targets that can be detected in the experiment; the range of X is 0-19.

[0047] Preferably, M is any value of 2, 3, 4, 5, 6, 7, or 8; more preferably, M is 2 or 3.

[0048] N can be 1, 2, or 3.

[0049] X is, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. Preferably, the range of X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. More preferably, the range of X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0050] The ratio of any two fluorescent reporter groups A and B ranges from 1:(0.25~10), for example, 1:0.25, 1:0.33, 1:0.75, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4. 1:4.6, 1:4.8, 1:5, 1:5.2, 1:5.4, 1:5.6, 1:5.8, 1:6, 1:6.2, 1:6.4, 1:6.6, 1:6.8, 1:7, 1:7.2, 1:7.4, 1:7.6, 1:7.8, 1:8, 1:8.2, 1:8.4, 1:8.6, 1:8.8, 1:9, 1:9.2, 1:9.4, 1:9.6, 1:9.8, 1:10, etc.;

[0051] Preferably, the ratio range of any two fluorescent reporter groups A and B is 1:0.25, 1:0.33, 1:0.75, 1:1, 1:2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, 1:5.2, 1:5.4, 1:5.6, 1:5.8, or 1:6.

[0052] In this application, A and B represent any fluorescent reporter group. For example, A can represent any one of FAM, VIC, ROX, Cy5, Cy5.5 and / or HEX, and B represents a fluorescent reporter group different from A, which can also be any one of FAM, VIC, ROX, Cy5, Cy5.5 and / or HEX.

[0053] More preferably, the ratio of the two fluorescent reporter groups is in the range of 4:1, 1:1, 1:3.2, or 1:4.

[0054] In one embodiment of this application, when M is 4, N is 2, and X is 10, the identified pathogen P is 10. In another embodiment of this application, when M is 5, N is 1, and X is 2, the identified pathogen P is 8.

[0055] In one specific approach, P probes are labeled using N concentrations of M different fluorescent groups. The difference in the type or concentration of the fluorescent group carried by each of the P probes means that the type of fluorescent group carried by each of the P probes is different, or that the concentration of the fluorescent group carried by each of the P probes is different, or that the type or concentration of the fluorescent group carried by each of the P probes is completely different. Those skilled in the art will understand that the core of this application lies in using the different types or fluorescence intensities of the fluorescent groups to distinguish each probe; as long as they can be distinguished and detected by detection methods, it is sufficient.

[0056] In one specific method, two fluorescent groups, A and B, are labeled on a first probe at a ratio of 1:0.33; on a second probe, they are labeled at a ratio of 1:0.75; on a third probe, they are labeled at a ratio of 1:1; on a fourth probe, they are labeled at a ratio of 1:2; and on a fifth probe, they are labeled at a ratio of 1:4. By adjusting the ratio of A and B fluorescent groups, the detection of different pathogens can be achieved.

[0057] In some implementations, the target gene can be any type of shrimp pathogen, including but not limited to the listed pathogens.

[0058] In some implementations, the target sequence is a variety of different shrimp pathogen sequences.

[0059] Preferably, the shrimp pathogens include 18 pathogenic microorganisms and virulence factors.

[0060] The combination of this application can effectively control the number of multiple probes and utilize various concentrations of multiple probes to specifically and efficiently detect a variety of pathogens in shrimp.

[0061] In one embodiment of this application, the above-mentioned primer and probe set is used to achieve simultaneous detection of 11 pathogens and internal controls in 1 well with 5-color fluorescence in 2 wells by digital PCR. The detection range covers the most common viruses, bacteria and parasite pathogens in shrimp farming.

[0062] The primers and probes of this application can be used in any PCR detection system for detecting target genes, including but not limited to conventional PCR, RL-PCR, RNA-PCR, quantitative real-time PCR, digital PCR, PCR-ELISA, nested PCR-high resolution melting analysis (nPCR-HRM), etc. Preferably, the primers and probes involved in this application are used for quantitative real-time PCR or digital PCR. Most preferably, the primers and probes determined in this application are used for digital PCR.

[0063] The digital PCR reaction premix in this application contains primers, probes, enzymes (hot-start Taq enzyme, reverse transcriptase, and / or UDG enzyme), dNTPs, glycerol, betaine, and NH4. + Mg 2+ Bovine serum albumin, etc.

[0064] Compared to the digital PCR reaction premix, the digital PCR amplification mixture in this application includes an amplification template (such as a sample template, positive control, or negative control) and distilled water. The positive control (also known as a positive quality control) can be a plasmid containing various target genes as described in Table 7; the negative control (also known as a negative quality control) uses enzyme-free sterile water as a template.

[0065] In some embodiments, this application provides a digital PCR reaction premix for detecting a target gene, the PCR reaction premix comprising the primer pairs described above, and combinations of primer pairs and probes for each pathogen described above.

[0066] In one specific embodiment, this application targets specific nucleotide sequences of 18 shrimp pathogens and virulence factors, namely PirA / B, WSSV, IMNV, VP, TSV, IHHNV, EHP, YHV, MBV, SHIV, HPV, VH, MrNV, XSV, CMNV, VA, SI, and PF. Through multiple sequence comparison and analysis, multiplex digital PCR primers and probes for detecting these pathogens and virulence factors were designed. By varying the concentration of the probes used, up to 11-12 multiplex digital PCR can be performed in a single well. Simultaneously, internal standard primers and probes were designed to monitor the sample collection and extraction process and avoid false negative results.

[0067] To improve the efficiency of screening for shrimp pathogens and virulence factors, this application provides a method for efficient screening of shrimp pathogens and virulence factors using PCR. Examples of upstream primers for PirA / B include SEQ ID NO:1 and downstream primers include SEQ ID NO:2; examples of upstream primers for WSSV include SEQ ID NO:4 and downstream primers include SEQ ID NO:5; examples of upstream primers for IMNV include SEQ ID NO:7 and downstream primers include SEQ ID NO:8; examples of upstream primers for VP include SEQ ID NO:10 and downstream primers include SEQ ID NO:11; examples of upstream primers for TSV include SEQ ID NO:13 and downstream primers include SEQ ID NO:14; examples of upstream primers for IHHNV include SEQ ID NO:16 and downstream primers include SEQ ID NO:17; and examples of upstream primers for EHP include SEQ ID NO:16. As shown in NO:19, an example of the downstream primer is shown in SEQ ID NO:20; an example of the upstream primer for GAPDH is shown in SEQ ID NO:22, and an example of the downstream primer is shown in SEQ ID NO:23; an example of the upstream primer for YHV is shown in SEQ ID NO:25, and an example of the downstream primer is shown in SEQ ID NO:26; an example of the upstream primer for MBV is shown in SEQ ID NO:28, and an example of the downstream primer is shown in SEQ ID NO:29; an example of the upstream primer for SHIV is shown in SEQ ID NO:31, and an example of the downstream primer is shown in SEQ ID NO:32; an example of the upstream primer for HPV is shown in SEQ ID NO:34, and an example of the downstream primer is shown in SEQ ID NO:35; an example of the upstream primer for VH is shown in SEQ ID NO:37, and an example of the downstream primer is shown in SEQ ID NO:38; an example of the upstream primer for MrNV is shown in SEQ ID NO:40, and an example of the downstream primer is shown in SEQ ID NO:29. As shown in NO:41; an example of an upstream primer for XSV is shown in SEQ ID NO:43, and an example of a downstream primer is shown in SEQ ID NO:44; an example of an upstream primer for CMNV is shown in SEQ ID NO:46, and an example of a downstream primer is shown in SEQ ID NO:47.An example of an upstream primer for VA is shown in SEQ ID NO:49, and an example of a downstream primer is shown in SEQ ID NO:50; an example of an upstream primer for SI is shown in SEQ ID NO:52, and an example of a downstream primer is shown in SEQ ID NO:53; an example of an upstream primer for PF is shown in SEQ ID NO:55, and an example of a downstream primer is shown in SEQ ID NO:56.

[0068] On the other hand, this application provides probes for screening the PirA / B gene, one example of which is shown in SEQ ID NO:3; probes for screening the WSSV gene, one example of which is shown in SEQ ID NO:6; probes for screening the IMNV gene, one example of which is shown in SEQ ID NO:9; probes for screening the VP gene, one example of which is shown in SEQ ID NO:12; probes for screening the TSV gene, one example of which is shown in SEQ ID NO:15; probes for screening the IHHNV gene, one example of which is shown in SEQ ID NO:18; probes for screening the EHP gene, one example of which is shown in SEQ ID NO:21; probes for screening the GAPDH gene, one example of which is shown in SEQ ID NO:24; probes for screening the YHV gene, one example of which is shown in SEQ ID NO:27; probes for screening the MBV gene, one example of which is shown in SEQ ID NO:30; and probes for screening the SHIV gene, one example of which is shown in SEQ ID NO:30. As shown in NO:33; a probe for screening HPV genes, one example of which is shown in SEQ ID NO:36; a probe for screening VH genes, one example of which is shown in SEQ ID NO:39; a probe for screening MrNV genes, one example of which is shown in SEQ ID NO:42; a probe for screening XSV genes, one example of which is shown in SEQ ID NO:45; a probe for screening CMNV genes, one example of which is shown in SEQ ID NO:48; a probe for screening VA genes, one example of which is shown in SEQ ID NO:51; a probe for screening SI genes, one example of which is shown in SEQ ID NO:54; a probe for screening PF genes, one example of which is shown in SEQ ID NO:57.

[0069] In one aspect, this application provides a combination of primers and probes capable of simultaneously screening 18 pathogenic microorganisms and virulence factors, wherein the combination of primers and probes comprises any one or more of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12; SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15; SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21; SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27; SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 ... NO: 31, SEQ ID NO: 32, SEQ ID NO: 33; SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42; SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45; SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48; SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51; SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54; SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57.

[0070] In some embodiments, the 5' end of the probe sequence used in this application is labeled with a fluorescent group, and the 3' end of the probe sequence used in this application is labeled with a fluorescence quencher group. When the above probes are in the same reaction system, the spectral range of the fluorescent groups used to screen different types of shrimp pathogens is different. The fluorescent group of the probe sequence used in this application can be any fluorescent group commonly used in the art, including but not limited to FAM, VIC, ROX, Cy5, Cy5.5 and / or HEX; the fluorescence quencher group of the probe sequence used in this application can be any fluorescence quencher group commonly used in the art, including but not limited to BHQ1, BHQ2, BHQ3, and / or Eclipse.

[0071] On the other hand, the probes used in this application and their 5' end fluorescent reporter groups and 3' end fluorescent quencher groups are as follows: the probe for screening PirA / B is shown in SEQ ID NO:3, with its 5' end fluorescent label being FAM and its 3' end fluorescent label being BHQ1, i.e., 5'-FAM-CATTTATGGCTGGCGCGCTGGAAAG-3'-BHQ1; the probe for screening WSSV is shown in SEQ ID NO:6, with its 5' end fluorescent label being FAM / HEX and its 3' end fluorescent label being BHQ1, i.e., 5'-FAM-TGGTATCCTCTTTCGCATTCGCCC-BHQ1-3' and 5'-HEX-TGGTATCCTCTTTCGCATTCGCCC-BHQ1-3'; the probe for screening IMNV is shown in SEQ ID NO:6. As shown in NO:9: its 5' end fluorescent label is HEX, and its 3' end fluorescent label is BHQ1, i.e., 5'-HEX-TACAATGTCGGAGCGCAGGAACAG-BHQ1-3'; the probe for screening VP is shown in SEQ ID NO:12, its 5' end fluorescent label is ROX, and its 3' end fluorescent label is BHQ2, i.e., 5'-ROX-TGGCGAACGAGAACGCAGACATTA-BHQ2-3'; the probe for screening TSV, one example of which is shown in SEQ ID NO:15, its 5' end fluorescent label is ROX / Cy5, and its 3' end fluorescent label is BHQ2, i.e., 5'-ROX-TCCTCACACAACAACGCATAA-BHQ2-3', 5'-Cy5-TCCTCACACAACAACGCATAA-BHQ2-3'; the probe for screening IHHNV is shown in SEQ ID As shown in NO:18, its 5' end fluorescent label is ROX / Cy5, and its 3' end fluorescent label is BHQ2, i.e., 5'-ROX-TGGTTTGTCCCACTTCATTTGGCC-BHQ2-3' and 5'-Cy5-TGGTTTGTCCCACTTCATTTGGCC-BHQ2-3'; the probe for screening EHP is shown in SEQ ID NO:21, its 5' end fluorescent label is Cy5, and its 3' end fluorescent label is BHQ2, i.e., 5'-Cy5-TGCATGGCCGTTGGAAATTGATGG-BHQ2-3'; the probe for screening GAPDH gene is shown in SEQ ID NO:24: its 5' end fluorescent label is Cy5.5, and its 3' end fluorescent label is BHQ3, i.e., 5'-Cy5.5-TGTGTTCACCACCATCGAGAAGGC-BHQ3-3';The probe for screening YHV is shown in SEQ ID NO:27, with its 5' end fluorescently labeled FAM and its 3' end fluorescently labeled BHQ1, 5'-FAM-CGTCTCACACACATGGACTTCACCT-BHQ1-3'; the probe for screening MBV is shown in SEQ ID NO:30, with its 5' end fluorescently labeled FAM / HEX and its 3' end fluorescently labeled BHQ1, namely 5'-FAM-TGCTGGGAATTTGCTAAAGTAACGACG-BHQ1-3' and 5'-HEX-TGCTGGGAATTTGCAAAGTAACGACG-BHQ1-3'; the probe for screening SHIV is shown in SEQ ID NO:27. As shown in NO:33: its 5' end fluorescent label is FAM / HEX, and its 3' end fluorescent label is BHQ1, i.e., 5'-FAM-ACGTTAAAGGGTCTCACGGGAAACG-BHQ1-3' and 5'-HEX-ACGTTAAAGGGTCTCACGGGAAACG-BHQ1-3'; the probe for screening HPV is shown in SEQ ID NO:36: its 5' end fluorescent label is FAM / HEX, and its 3' end fluorescent label is BHQ1, i.e., 5'-FAM-ACTGTTCATCGTATCTTCCGCCGC-BHQ1-3' and 5'-HEX-ACTGTTCATCGTATCTTCCGCCGC-BHQ1-3'; the probe for screening VH is shown in SEQ ID NO:36. As shown in NO:39, its 5' end fluorescent label is HEX, and its 3' end fluorescent label is BHQ1, i.e., 5'-HEX-CGCATAATACCTTCGGGTCAAAGAGGG-BHQ1-3'; the probe for screening MrNV is shown in SEQ ID NO:42, its 5' end fluorescent label is ROX, and its 3' end fluorescent label is BHQ2, i.e., 5'-ROX-CCTGGTAGTTCCCGAAGCGAATGT-BHQ2-3'; the probe for screening XSV is shown in SEQ ID NO:45, its 5' end fluorescent label is ROX / Cy5, and its 3' end fluorescent label is BHQ2, i.e., 5'-ROX-CCATGATCCTCGCATCGTATCGCA-BHQ2-3', 5'-Cy5-CCATGATCCTCGCATCGTATCGCA-BHQ2-3'; the probe for screening CMNV is shown in SEQ ID NO:45. As shown in NO:48, its 5' end fluorescent label is ROX / Cy5, and its 3' end fluorescent label is BHQ2, namely 5'-ROX-TTGTATGGTGGAGCTGCGTCAAGT-3' and 5'-Cy5-TTGTATGGTGGAGCTGCGTCAAGT-BHQ2-3'.The probe for screening VA is shown in SEQ ID NO:51, with its 5' end fluorescently labeled ROX / Cy5 and its 3' end fluorescently labeled BHQ2, i.e., 5'-ROX-ACACAACAACCATTGTGGGTGGC-BHQ2-3' and 5'-Cy5-ACACAACAACCATTGTGGGTGGC-BHQ2-3'; the probe for screening the SI gene is shown in SEQ ID NO:54, with its 5' end fluorescently labeled Cy5 and its 3' end fluorescently labeled BHQ2, i.e., 5'-Cy5-TGCAGTTAAAGGGTCCGTAGTCGT-BHQ2-3'; the probe for screening the PF gene is shown in SEQ ID NO:57, with its 5' end fluorescently labeled Cy5.5 and its 3' end fluorescently labeled BHQ3, i.e., 5'Cy5.5-ATCCACTTCAGCTGGGACATCCTG-BHQ3-3'.

[0072] In some embodiments, the digital PCR reaction premix provided in this application may, in addition to the primer pairs described above, or any combination of primer pairs and probes described above, also include any four, five, six, seven, eight, or nine components selected from the following: hot-start Taq enzyme, reverse transcriptase, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, and NH4. + and / or Mg 2+ ion.

[0073] This application provides a kit for detecting a target gene, comprising the primer pairs described above, or any combination of primer pairs and probes described above, or a combination of the primer pairs described above and any probes. The kit of this application may also include any four, five, six, seven, eight, or nine components selected from the following:

[0074] Hot-start Taq enzyme, reverse transcriptase, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + and Mg 2+ ion.

[0075] The kits in this application may also include positive controls and / or negative controls.

[0076] In some embodiments, this application provides a method for detecting shrimp pathogens and virulence factors for non-disease diagnosis purposes, the method comprising the steps of:

[0077] (1) Processing the sample to be tested;

[0078] Use 100-1000 μL of sample for testing, preferably 100-200 μL, 600-1000 μL, 700-1000 μL, 800-1000 μL, or 900-1000 μL, with 1000 μL being the most preferred for improving detection rate. Centrifuge at 800 rpm for 10 min and collect the supernatant. The separated sample can be stored at -20℃±5℃ for 3 years. It is recommended to use a commercially available kit to extract total nucleic acids, including DNA and RNA, from the sample. After nucleic acid extraction, it is advisable to perform testing immediately; otherwise, it is best to store at -20℃ or below for no more than 3 months.

[0079] (2) Prepare the digital PCR amplification mixture according to Table 1, which contains the sample to be tested provided in step (1) and the digital PCR reaction premix below; the final concentrations of the upstream and downstream primers in the digital PCR reaction premix are 50-900 nM, preferably 50-500 nM, 200-500 nM, and most preferably 450 nM, respectively; the final concentrations of each probe are 50-800 nM, preferably 50-500 nM, 100-400 nM, and most preferably 250 nM, respectively; Mg 2+ The final concentration of the ions is 0.5-5 mM, preferably 1-4.5 mM, 1.5-4 mM, 2-3.5 mM, and most preferably 3 mM; the final concentration of bovine serum albumin is 0.01-1 wt%, preferably 0.02-0.5 wt%, 0.03-0.1 wt%, 0.04-0.08 wt%, and most preferably 0.05%; the final concentration of the hot-start Taq enzyme (which can be antibody-modified hot-start Taq enzyme, chemically modified hot-start Taq enzyme, or aptamer-modified hot-start Taq enzyme) is 0.5-10 U / reaction, preferably 1-5 U / freaction, 1.5-4 U / reaction, 2-3.5 U / reaction, and most preferably 2.5 U / reaction; the final concentration of the reverse transcriptase is 0.5-10 U / reaction, preferably 1-5 U / reaction, 1.5-4 U / reaction, 2-3.5 U / reaction, and most preferably 2.5 U / reaction. The final concentration of dNTPs is 20-300 µM, preferably 50-250 µM, 100-230 µM, 150-220 µM, and most preferably 200 µM. The digital PCR reaction premix may also contain UNG enzyme at a final concentration of 0.1-10 U / reaction, 0.5-5 U / reaction, 1-2.5 U / reaction, 1-2 U / reaction, and most preferably 1.5 U / reaction. The pH of the digital PCR reaction premix is ​​7-9, preferably pH 7.4-8.5, preferably pH 7.8-8.2, and most preferably pH 8.0.

[0080] Those skilled in the art will understand that the substances and concentrations listed in the above PCR amplification mixture are illustrative, and can be selected based on the actual system.

[0081] Table 1. Digital PCR amplification mixture

[0082]

[0083] (3) Prepare microdroplets and perform PCR amplification reaction;

[0084] Step 1: Preparation of microdroplets. First, add 50-100 μl of droplet-generating oil into the square filling hole at the top of the microfluidic cartridge, preferably 70-100 μl, and most preferably 75 μl.

[0085] Step 2: Add 13-25 μl of digital PCR amplification mixture (containing amplified nucleic acid template, primers, probes, etc.) into the circular sample well at the top of the microfluidic cartridge, preferably 15-25 μl, and most preferably 20 μl;

[0086] Step 3: Cover the top sample loading port and oil filling port of the microfluidic cartridge, and the bottom waste liquid port with two sealing caps; place the microfluidic cartridge containing the PCR amplification mixture on a PCR amplification instrument, preferably an SG-2000 PCR amplification instrument. The amplification reaction can be performed according to the amplification program recommended in Table 2:

[0087] Table 2. Reverse transcription and PCR amplification procedures

[0088]

[0089] (4) Signal collection

[0090] On a biochip reader (e.g., Dscanner4-1000, registration certificate number: Su Medical Device Registration 20202220818), select DQ mode and set the fluorescence channels to FAM, HEX, ROX, Cy5, and Cy5.5. Amplify the high-throughput digital PCR cartridge (e.g., microfluidic cartridge) from a PCR amplification instrument, preferably an SG-2000 PCR amplification instrument (registration certificate number: Su Medical Device Registration 20202220887). After the reaction is complete, remove the cartridge and transfer it smoothly to the biochip reader for signal collection.

[0091] (5) Results Analysis

[0092] Click "Analyze" on the biochip reader to analyze the experimental data. First, check if the "Accepted Droplets" count is ≥10000 to proceed with further analysis. Click "2D Amplitude" to view the 2D scatter plots for channels TAM, HEX, ROX, and Cy5. Based on the positive control, click the 2D scatter plot to divide the positive droplets into different regions. In the 2D scatter plot with FAM as the Y-axis and HEX as the x-axis (e.g.,...),... Figure 1D The darkest black dot represents a droplet without amplified template; the light blue dot represents a droplet at position 1 containing a FAM-labeled probe; the green dot represents a droplet at position 2 containing both FAM and HEX-labeled probes; the orange dot represents a droplet at position 3 containing both FAM and HEX-labeled probes; the magenta dot represents a droplet at position 4 containing both FAM and HEX-labeled probes; and the brownish-gray dot represents a droplet at position 5 containing a HEX-labeled probe. This is shown in a 2D scatter plot with ROX as the Y-axis and Cy5 as the x-axis (e.g., Figure 1E The darkest black dot represents a droplet without amplified template; the light blue dot represents a droplet at position 1 containing a ROC-labeled probe; the green dot represents a droplet at position 2 containing both ROX and Cy5-labeled probes; the orange dot represents a droplet at position 3 containing both ROX and Cy5-labeled probes; the magenta dot represents a droplet at position 4 containing both ROX and Cy5 probes; and the brownish-gray dot represents a droplet at position 5 containing a Cy5 probe. View the Cy5.5 channel droplets in "1D Amplitude".

[0093] The primers, probes, digital PCR reaction premixes containing primers and probes, kits, and microfluidic cartridges provided in this application can all be used to prepare quantitative detection kits for detecting multiple pathogens in shrimp tissues. They can also be used alone or in combination with other reagents for the detection of multiple pathogens in shrimp tissues. The processing steps for shrimp tissue samples are as follows: Take 100-1000 μL of the shrimp tissue sample to be tested, preferably 100-200 μL, 600-1000 μL, 700-1000 μL, 800-1000 μL, or 900-1000 μL, with 1000 μL being the most preferred to improve the detection rate. Use tissue homogenizer to homogenize the shrimp tissue into a paste. Take 200 μL of the shrimp paste for nucleic acid extraction. It is recommended to use a commercially available kit to extract microbial nucleic acids from the shrimp paste sample. After nucleic acid extraction, it is recommended to perform the test immediately; otherwise, it is best to store it below -20℃ for no more than 3 months.

[0094] In some implementations, in addition to shrimp tissue, the types of samples involved in the detection also include shrimp feed, shrimp farming water, and / or soil.

[0095] For convenience, the sequences of primers and probes used in this application are summarized in the table below:

[0096] Table 3. Primer and probe sequences of this application targeting 18 microbial pathogens, virulence factors, and internal reference genes.

[0097] ;

[0098] .

[0099] Table 4. Examples of probe fluorescent labels used in this application

[0100]

[0101] Example

[0102] Example 1. Detection kit for multiple pathogens in shrimp pathogen infection

[0103] This application develops a digital PCR detection kit for detecting 18 pathogens and virulence factors plus one internal reference gene in shrimp tissues. The kit contains primer and probe mixture 1, primer and probe mixture 2, digital PCR premix (digital PCR buffer, dNTPs, MgCl2, Taq hot-start enzyme mixture, reverse transcriptase, glycerol, betaine), negative control, and positive control (examples of specific positive and negative control samples are shown in Table 7).

[0104] Each sample requires three sets of tests. Using the primer-probe mixture and digital PCR premix provided in the kit, digital PCR droplet generation, reverse transcription, and PCR amplification are performed on the sample. The primer-probe mixture volume is 2 μL, the digital PCR premix volume is 10 μL, the DNA loading volume is 8 μL, and the total reaction volume is 20 μL. The reaction system for digital PCR amplification is shown in Table 5-6.

[0105] Table 5: Group 1 Multiple Numerical Reaction System

[0106]

[0107] Table 6: Group 2 Multiple Numerical Reaction Systems

[0108]

[0109] The components of the kit in this application are shown in Table 7.

[0110] Table 7: Components of the Reagent Kit

[0111]

[0112] The kit of this application includes an internal quality control system to monitor the sample collection and extraction process and avoid false negatives. The kit also includes negative and positive quality controls (examples of which are shown in Table 7) to monitor digital PCR amplification and microarray reading, and to assist in the interpretation of the final results. The specific steps are as follows:

[0113] 1) Release the nucleic acid from the sample to be tested;

[0114] 2) In one well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, add a mixture of primers and fluorescently labeled probes for 7 pathogens, virulence factors (PirA / B) and internal control (GAPDH) (corresponding to group 1, see Table 7) and digital PCR buffer;

[0115] 3) In another well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, add a mixture of primers and fluorescently labeled probes for another 11 pathogens (corresponding to group 2, see Table 7) and digital PCR buffer;

[0116] 4) Perform digital PCR droplet generation, reverse transcription, and PCR amplification in a digital PCR amplification instrument; add 75 μl of oil-phase-coated reagent to the oil wells of the high-throughput digital PCR chip, add 20 μl of amplification mixture to the reagent wells, cover with a sealing cap, and place on a PCR amplification instrument to perform droplet generation, reverse transcription, and PCR amplification.

[0117] Perform reverse transcription and PCR amplification reactions according to the following recommended amplification procedures:

[0118] Table 8. Reverse transcription and PCR amplification procedures

[0119]

[0120] 5) Signal collection

[0121] Using the nucleic acid obtained in step 4) as a template, the chip is used as the detection material after amplification. Fluorescence is detected by a biochip reader, and the results are interpreted by the presence and intensity of fluorescence in each droplet.

[0122] Result interpretation:

[0123] The results are shown in Figure 1 and Table 9. The first well detected 7 pathogenic microorganisms and virulence factors, and the second well detected 11 pathogens.

[0124] Table 9. Pathogens and virulence factors corresponding to different positions in Well 1 and Well 2

[0125]

[0126] If, in a 2D scatter plot, there are positive droplets in the positive droplet region, excluding negative droplets, and the number of droplets is greater than the threshold, then the test result of the strain corresponding to that location is determined to be positive.

[0127] Table 10. Pathogens and virulence factors corresponding to positive clusters in shrimp

[0128]

[0129] Example 2. Optimizing the detection efficiency of different primer probes for pathogens

[0130] For each shrimp pathogen, 2-4 sets of primers and probes were designed for detection. The primer and probe pairs used in the experiment are shown in Tables 3 and 12. For each pathogen, primer and probe mixtures (2-4 sets of primers and probes were mixed) were prepared using the primers and probes in Tables 3 and 12, digital PCR premix (digital PCR buffer, dNTPs, MgCl2, Taq hot-start enzyme mixture, reverse transcriptase, glycerol, betaine), and corresponding positive control samples for the pathogen targeted by the primers and probes (see Table 7 for examples of specific positive and negative control samples).

[0131] Each primer and probe kit for each pathogen requires two sets of detections. Using the primer-probe mixture and digital PCR premix provided in the kit, digital PCR droplet generation, reverse transcription, and PCR amplification are performed on the test samples. The primer-probe mixture volume is 2 μL, the digital PCR premix volume is 10 μL, the DNA sample volume is 8 μL, and the total reaction volume is 20 μL. The reaction system for digital PCR amplification is shown in Table 5-6, and the remaining operations are the same as in Example 1.

[0132] Table 11: Digital reaction system for primer and probe optimization

[0133]

[0134] Set 1 refers to the primers and probes described in Table 3, and sets 2-4 refer to the primers and probes described in Table 12.

[0135] Table 12. Primer and probe sequences used in this application for optimization of 18 microbial pathogens, virulence factors, and internal reference genes.

[0136] ;

[0137] .

[0138] By screening different primer-probe pairs for each pathogen or virulence factor, the primer-probe pair with the highest detection efficiency was selected. The selected primer-probe pairs are shown in Table 3. Figures 2A-2C The results of testing for three of the pathogens are listed in the document. Figure 2A The results were obtained by amplifying PirA / B positive plasmids with the same concentration and volume using four sets of PirA / B primers and probes. PirA / B-1 (i.e., PirA / B-f1, PirA / B-r1 and PirA / B-P1 as described in Table 3) had the highest copy number, so this set of primers and probes was selected for use in the kit. Figure 2B The results were obtained by amplifying WSSV positive plasmids with the same concentration and volume using four sets of WSSV primers and probes. WSSV-1 (i.e., WSSV-f1, WSSV-r1 and WSSV-P1 as described in Table 3) had the highest copy number, so this set of primers and probes was selected for use in the kit. Figure 2C The results were obtained by amplifying IMNV positive plasmids with the same concentration and volume using four sets of IMNV primers and probes. IMNV-1 (i.e., IMNV-f1, IMNV-r1 and IMNV-P1 as described in Table 3) had the highest copy number, so this set of primers and probes was selected for use in the kit.

[0139] Example 3. Specificity Detection

[0140] This application selects *Porphyromonas violaceum*, *Streptococcus intermedia*, *Bordetella bronchiseptica*, *Serratia marcescens*, *Streptococcus distantly*, *Listeria esculenta*, *Burkholderia cepacia*, *Streptococcus pharyngitis*, *Streptococcus anaerobicans*, *Streptococcus sanguinis*, *Streptococcus constellus* subspecies, *Veillonella spp.*, *Streptococcus agalactiae*, *Listeria spp.*, *Corynebacterium bandingense*, *Aeromonas tempera*, *Streptococcus salivarius*, *Aeromonas guinea*, *Klebsiella terrestriala*, *Staphylococcus capitulata* subspecies, *Streptococcus galactiae*, *Micrococcus luteus*, *Shigella sonnei*, *Staphylococcus aureus*, *Listeria spp.*, *Rahn's bacillus*, *Salmonella typhimurium*, *Lactobacillus acidophilus*, and *Aeromonas hydrophila*. *Streptococcus faecalis*, *Legionella pneumophila* subsp. *spareunia*, *Staphylococcus schlegelii* subsp. *schlegelii*, *Pseudomonas schlegelii*, *Enterobacter carcinogenae*, *Sphingosine monocytogenes*, *Neisseria lactis*, *Staphylococcus carcinoma*, *Vibrio parahaemolyticus*, *Streptococcus canis*, *Enterococcus pyogenes*, *Proteus mirabilis*, *Pseudomonas aeruginosa*, *Bacteroides vulgaris*, *Acinetobacter p. p.*, *R. p. p.*, *Streptococcus bovis*, *Eikenella vesicola*, *Streptococcus pyogenes*, *Neisseria meningitidis*, *Enterococcus staphylococcus xylose*, *Moraxella catarrhalis*, *Morganella morganii*, *Pseudomonas mendoza*, *Enterococcus cecum*, *Streptococcus equi* subsp. *porphyria*, *Rhodococcus equi*, *Basilella marneffei*, *Twincocephalus measlese*, *Bacteroides ovalis* Staphylococcus rudoidea, Acinetobacter rudoidea, Haemophilus influenzae, Neisseria gonorrhoeae, Shigella dysenteriae, Shigella-like bacteria, Bacillus cereus, Bacillus subtilis, Streptococcus stomatitis, Acinetobacter radiata, Enterobacter cobesii, Agrobacterium, Streptococcus gallate, Staphylococcus pseudointermediate, Enterobacter hominis, Streptococcus pyogenes, Enterococcus haematobacterium, Listeria glabrata, Streptococcus gordonii, Neisseria spp., Streptococcus parahaemolyticus, Clostridium difficile, Bordetella parapertussis, Staphylococcus saprophyticus, Shigella flexneri, Yersinia freundii, Haffnerella vesicola, non-communicable luminescent bacteria, Staphylococcus aureus, Pseudomonas putida, Bacteroides polymorpha, Burkholderia polyphaga Specific reactions were performed on 115 strains of bacteria, including *Dryobacterium tumefaciens*, *Pasteurella multocida*, *Pseudomonas dell'sae*, *Bacillus licheniformis*, *Bacteroides monomorpha*, *Listeria monocytogenes*, *Propionibacterium acnes*, *Vibrio vulnificus*, *Pantotheca cum Bile*, *Salmonella enteritidis*, *Yersinia enterocolitica*, *Klebsiella pneumoniae*, *Pseudomonas alkaligenes*, *Streptococcus mutans*, *Animalobacterium berezig*, *Acinetobacter berezii*, *Shigella boydii*, *Actinobacter actinomycetii*, *Cronobacter sakazakii*, *Bordeia pertussis*, *Enterococcus fowleri*, *Clostridium perfringens*, *Staphylococcus aureus*, and *Haemophilus parainfluenzae*. These strains are common clinical pathogens that easily cause clinical symptoms. All of the above strains were confirmed to be undetectable.

[0141] (1) Release the nucleic acid of the test strain;

[0142] (2) Mix the nucleic acids extracted using the above-mentioned strains;

[0143] (3) Digital PCR droplet formation and amplification:

[0144] Digital PCR droplet formation and digital PCR amplification were performed using the reaction system and procedure described in Example 1.

[0145] (4) Signal collection:

[0146] The digital PCR chip was removed from the digital PCR amplification instrument and smoothly transferred to a biochip reader for signal collection.

[0147] (5) Result interpretation and analysis:

[0148] A threshold line is drawn based on the midpoint between the negative and positive droplets in the positive control sample of the kit. If the number of positive droplets in the mixed sample is greater than the threshold, the test result of the bacterial mixture corresponding to that positive droplet is considered positive. The software displays the copy number corresponding to the positive droplet. A linear regression plot is constructed with dilution on the x-axis and the measured value log for each dilution on the y-axis.

[0149] The results of cross-reactivity testing of 115 strains are as follows: Figure 3 As shown, no cross-reaction was detected between these 115 strains and the primers and probes for the 18 pathogens in the kit. Figure 3 Only negative droplets were detected; no positive droplets were found to cross-react with the 115 strains. This indicates that the primers and probes described in this application have good detection specificity.

[0150] Example 4. Detection of samples carrying drug-resistant gene strains using the detection kit

[0151] The method of Example 1 was used to test the samples carrying shrimp pathogens. The diseased shrimp samples came from a shrimp pond in Nantong. Figure 4A This indicates the detection results for WSSV virus, showing that the WSSV virus gene was detected in the shrimp tissue sample; Figure 4B The result of the VP test indicates that VP bacteria were detected in the shrimp tissue sample tested. Figure 4C A positive result for the detection of the PirA / B virulence gene indicates that the PirA / B virulence factor was detected in the sample.

[0152] Example 5. Detection of pathogens in shrimp feed samples using the detection kit.

[0153] The method described in Example 1 was used to test 14 samples, including water, soil, shrimp feed, and shrimp tissue, obtained from a shrimp farming base in a certain area.

[0154] The results showed that VSSV virus was detected in one sample. Figure 5 ).

[0155] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A primer and probe combination for ultramultiplex digital PCR for the specific detection of target genes of multiple pathogens in shrimp, wherein the primers and probes consist of the following two sets of primers and probes, wherein: The first set of primers and probes is a combination of PCR primers and probes for specifically amplifying a specific fragment of the virulence factor PirA / B. The primers include an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:

2. The probes include a probe as shown in SEQ ID NO:

3. A PCR primer and probe combination for specifically amplifying a specific fragment of WSSV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5, and the probes include a probe as shown in SEQ ID NO:6; A PCR primer and probe combination for specifically amplifying a specific fragment of the IMNV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:7 and a downstream primer as shown in SEQ ID NO:8, and the probes include a probe as shown in SEQ ID NO:9; A PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio parahaemolyticus VP, wherein the primers include an upstream primer as shown in SEQ ID NO:10 and a downstream primer as shown in SEQ ID NO:11, and the probe includes a probe as shown in SEQ ID NO:12; A PCR primer and probe combination for specifically amplifying a specific fragment of TSV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:13 and a downstream primer as shown in SEQ ID NO:14, and the probes include a probe as shown in SEQ ID NO:15; A PCR primer and probe combination for specifically amplifying a specific fragment of the IHHNV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:16 and a downstream primer as shown in SEQ ID NO:17, and the probes include a probe as shown in SEQ ID NO:18; A PCR primer and probe combination for specifically amplifying a specific fragment of EHP from shrimp hepatocellular carcinoma, wherein the primers include an upstream primer as shown in SEQ ID NO:19 and a downstream primer as shown in SEQ ID NO:20, and the probe includes a probe as shown in SEQ ID NO:21; A PCR primer and probe combination for specifically amplifying a specific fragment of the internal reference gene GAPDH, wherein the primers include an upstream primer as shown in SEQ ID NO:22 and a downstream primer as shown in SEQ ID NO:23, and the probe includes a probe as shown in SEQ ID NO:24; The second set of primers and probes consists of a combination of PCR primers and probes for specifically amplifying specific fragments of the YHV virus. The primers include an upstream primer as shown in SEQ ID NO:25 and a downstream primer as shown in SEQ ID NO:

26. The probes include a probe as shown in SEQ ID NO:

27. A combination of PCR primers and probes for specifically amplifying specific fragments of MBV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:28 and a downstream primer as shown in SEQ ID NO:29, and the probes include a probe as shown in SEQ ID NO:30; A PCR primer and probe combination for specifically amplifying a specific fragment of SHIV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:31 and a downstream primer as shown in SEQ ID NO:32, and the probes include a probe as shown in SEQ ID NO:33; A PCR primer and probe combination for specifically amplifying a specific fragment of HPV virus, wherein the primers comprise an upstream primer as shown in SEQ ID NO:34 and a downstream primer as shown in SEQ ID NO:35, and the probe comprises a probe as shown in SEQ ID NO:36; and a PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio harveyi VH, wherein the primers comprise an upstream primer as shown in SEQ ID NO:37 and a downstream primer as shown in SEQ ID NO:38, and the probe comprises a probe as shown in SEQ ID NO:

39. A PCR primer and probe combination for specifically amplifying a specific fragment of MrNV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:40 and a downstream primer as shown in SEQ ID NO:41, and the probes include a probe as shown in SEQ ID NO:42; A PCR primer and probe combination for specifically amplifying a specific fragment of the XSV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:43 and a downstream primer as shown in SEQ ID NO:44, and the probes include a probe as shown in SEQ ID NO:45; A PCR primer and probe combination for specifically amplifying a specific fragment of CMNV virus, wherein the primers include an upstream primer as shown in SEQ ID NO:46 and a downstream primer as shown in SEQ ID NO:47, and the probes include a probe as shown in SEQ ID NO:48; A PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio alginolyticus VA, wherein the primers include an upstream primer as shown in SEQ ID NO:49 and a downstream primer as shown in SEQ ID NO:50, and the probe includes a probe as shown in SEQ ID NO:51; A PCR primer and probe combination for specifically amplifying a specific fragment of *Pseudomonas fluorescens* PF, wherein the primers comprise an upstream primer as shown in SEQ ID NO:55 and a downstream primer as shown in SEQ ID NO:56, and the probe comprises a probe as shown in SEQ ID NO:

57.

2. The primer and probe combination for ultramultiplex digital PCR according to claim 1, wherein the 5' end of the probe sequence is labeled with a fluorescent reporter group and the 3' end of the probe sequence is labeled with a fluorescent quencher group; when the probes are located in the same reaction system, the spectral range of the fluorescent reporter groups for screening different target genes is different.

3. The primer and probe combination for ultramultiplex digital PCR according to claim 2, wherein the fluorescent reporter group is selected from FAM, VIC, ROX, Cy5, Cy5.5 and / or HEX; and the fluorescent quencher group is selected from BHQ1, BHQ2, BHQ3 and / or Eclipse.

4. The primer and probe combination for ultramultiplex digital PCR according to claim 3, wherein the probes used and their 5' end-labeled fluorescent reporter groups and 3' end-labeled fluorescent quencher groups are as follows: The probes for screening PirA / B are shown in SEQ ID NO:3, with FAM as the 5' end fluorescent label and BHQ1 as the 3' end fluorescent label; the probes for screening WSSV are shown in SEQ ID NO:6, with FAM / HEX as the 5' end fluorescent label and BHQ1 as the 3' end fluorescent label; the probes for screening IMNV are shown in SEQ ID NO:9, with HEX as the 5' end fluorescent label and BHQ1 as the 3' end fluorescent label; the probes for screening VP are shown in SEQ ID NO:12, with ROX as the 5' end fluorescent label and BHQ2 as the 3' end fluorescent label; the probes for screening TSV are shown in SEQ ID NO:15, with ROX / Cy5 as the 5' end fluorescent label and BHQ2 as the 3' end fluorescent label; the probes for screening IHHNV are shown in SEQ ID NO:18, with ROX / Cy5 as the 5' end fluorescent label and BHQ2 as the 3' end fluorescent label; the probes for screening EHP are shown in SEQ ID NO:

18. As shown in SEQ ID NO:21, its 5' end fluorescent label is Cy5, and its 3' end fluorescent label is BHQ2; the probe for screening the internal control GAPDH is shown in SEQ ID NO:24, its 5' end fluorescent label is Cy5.5, and its 3' end fluorescent label is BHQ3; the probe for screening YHV is shown in SEQ ID NO:27, its 5' end fluorescent label is FAM, and its 3' end fluorescent label is BHQ1; the probe for screening MBV is shown in SEQ ID NO:30, its 5' end fluorescent label is FAM / HEX, and its 3' end fluorescent label is BHQ1; the probe for screening SHIV is shown in SEQ ID NO:33, its 5' end fluorescent label is FAM / HEX, and its 3' end fluorescent label is BHQ1; the probe for screening HPV is shown in SEQ ID NO:36, its 5' end fluorescent label is FAM / HEX, and its 3' end fluorescent label is BHQ1; the probe for screening VH is shown in SEQ ID NO:

24. As shown in SEQ ID NO:39, its 5' end fluorescent label is HEX and its 3' end fluorescent label is BHQ1; the probe for screening MrNV is shown in SEQ ID NO:42, its 5' end fluorescent label is ROX and its 3' end fluorescent label is BHQ2; the probe for screening XSV is shown in SEQ ID NO:45, its 5' end fluorescent label is ROX / Cy5 and its 3' end fluorescent label is BHQ2; the probe for screening CMNV is shown in SEQ ID NO:48, its 5' end fluorescent label is ROX / Cy5 and its 3' end fluorescent label is BHQ2; the probe for screening VA is shown in SEQ ID NO:51, its 5' end fluorescent label is ROX / Cy5 and its 3' end fluorescent label is BHQ2; the probe for screening PF is shown in SEQ ID NO:57, its 5' end fluorescent label is Cy5.5 and its 3' end fluorescent label is BHQ3.

5. The combination according to any one of claims 1-4, wherein it is a combination used for digital PCR (dPCR) reaction.

6. A multi-multiplex digital PCR reaction premix for specific detection of a target gene, wherein the digital PCR reaction premix comprises a combination according to any one of claims 1-5.

7. The premixed solution for ultramultiplex digital PCR reaction according to claim 6, wherein the premixed solution further comprises four, five, six, seven, eight, or nine components selected from the following: hot-start Taq enzyme, reverse transcriptase, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, and NH4+. + and Mg 2+ ion.

8. A multi-level digital PCR microfluidic cartridge, wherein the digital PCR microfluidic cartridge comprises the multi-level digital PCR reaction premix according to claim 6 or claim 7.

9. The microfluidic cartridge according to claim 8, wherein the microfluidic cartridge includes at least one aperture.

10. The microfluidic cartridge according to claim 9, wherein the microfluidic cartridge includes at least two holes.

11. A kit for detecting a target gene, wherein the kit comprises a multi-multiplex digital PCR reaction premix for specifically detecting the target gene as described in claim 6 or claim 7.

12. The kit according to claim 11, wherein the kit further comprises a positive control, a negative control, and / or a droplet-generating oil.

13. The kit of claim 12, wherein the kit further comprises the ultramultiplex digital PCR microfluidic cartridge of claim 9 or 10.

14. A method for detecting a target gene for non-disease diagnosis purposes, the method comprising the following steps: (1) Release the nucleic acid from the sample to be tested; (2) Prepare a digital PCR amplification mixture containing the nucleic acid of the sample to be tested provided in step (1), the combination according to any one of claims 1-5, and / or the ultra-multiplex digital PCR reaction premix according to claim 6 or 7; (3) Prepare microdroplets in a multi-multiplex digital PCR microfluidic cartridge and perform PCR amplification reaction; (4) Use a multi-multiplex digital PCR microfluidic cartridge reader to read the fluorescence signal after the droplet PCR amplification reaction.

15. The method for detecting target genes for non-disease diagnosis according to claim 14, wherein the sample to be tested is selected from water, soil, shrimp feed and / or shrimp tissue from a shrimp farming base.

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