Super-multiple digital PCR (polymerase chain reaction) detection method for simultaneously detecting multiple pathogens of shrimps and application of super-multiple digital PCR detection method

Abstract

The invention relates to a super-multiplex digital PCR (polymerase chain reaction) detection method for simultaneously detecting multiple pathogens of shrimps and application of the super-multiplex digital PCR detection method. According to the digital PCR primer, the probe, the kit and the detection method for shrimp pathogen detection, pathogens aiming at 18 shrimp pathogens can be rapidly, accurately and ultrasensitively detected. By detecting pathogens in the prawns, the health condition of the prawns in intensive prawn culture can be evaluated in time, disease transmission can be prevented, intervention measures can be taken in time, the risk of disease transmission to prawn groups in intensive culture can be reduced, serious economic loss can be avoided, and the application value is extremely high. Accurate identification of specific pathogens in a shrimp sample is very important for designing a proper control strategy, because each pathogen may require different prevention and management methods, drug use conditions can be effectively reduced. The shrimp pathogen detection can improve the culture benefit, and is also beneficial for developing the epidemic disease prevention and control work of aquatic animals and the inspection and quarantine monitoring plan of main aquaculture variety offspring seeds.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to primers, probes, methods and kits for super-multiplex digital PCR for simultaneously detecting multiple pathogens of shrimp. Background Art

[0002] Shrimp pathogens are bacteria, viruses, parasites and protozoa. Viruses are the most important pathogenic factors of 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 to be the most serious of the approximately 20 viral pathogens of shrimp. It is also one of the main viral pathogens that have harmed artificially cultured shrimp in my country and the Asia-Pacific region in recent years. It can infect most types of shrimp with a high mortality rate. In addition, it can infect a variety of crabs, lobsters, amphipods, water flies and other crustaceans in freshwater and marine ecosystems. It has a wide host range and causes serious economic losses to the aquaculture industry. ② Another DNA virus is the infectious hypodermal and hematopoietic necrosis virus IHHNV, which is the smallest virus known among shrimp viruses and an important viral pathogen of shrimp, causing serious economic losses to the shrimp farming industry (up to 50% of the overall economic losses of the shrimp farming industry). IHHNV is a single-stranded DNA virus. IHHNV can cause poor growth, irregular growth and epidermal deformity of shrimp at harvest. The disease caused is infectious subcutaneous and hematopoietic necrosis, which is listed as an important crustacean disease that must be reported to the World Organization for Animal Health (OIE). ③ Penaeus monodon baculovirus MBV. ④ Shrimp hemocyte iridovirus SHIV is a newly discovered iridovirus that infects aquatic crustaceans. SHIV can be transmitted horizontally through feces, cannibalism, etc. Vannamei shrimp infected with SHIV will show clinical symptoms of anorexia, gastrointestinal tract emptying, hepatopancreas color change and death. SHIV is widely prevalent in China's main shrimp farming areas. It not only infects white shrimp, but also Macrobrachium rosenbergii, Penaeus monodon, Japanese shrimp, and green shrimp. ⑤ Shrimp hepatopancreatic parvovirus HPV. After being infected with HPV, the larvae are inactive, have a decreased appetite, grow slowly, rarely molt, and often have dirt hanging on their body surfaces, or many commensal organisms are fixed. During the breeding period, young or adult shrimps are thin, dark in color, and have a lot of black spots on the surface of their shells. Some of their shells become soft and their abdominal muscles turn white. They have poor resistance to adversity 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, discoloration of the tail, reduction in the size of the hepatopancreas, and gradual necrosis of the tail fan. Since there is no effective drug or vaccine for IMNV, sensitive and reliable diagnosis is required to take appropriate control measures, so prevention, management and timely diagnosis are the most effective means. ② Yellow head virus YHV, which causes yellow head disease (YHD), is a single-stranded RNA virus, and the envelope glycoprotein (gp116) has been shown to be the main virulence factor of YHV genotype 1. Acute infection of shrimp will cause symptoms such as stopping feeding within 2 to 4 days, with a high mortality rate. The head and chest of dying shrimp turn yellow due to the yellowing of the hepatopancreas, so it is called yellow head disease. my country has listed it as a Class II epidemic disease, and OIE has listed it as a disease that must be reported. ③ Taura syndrome virus TSV is one of the three most serious shrimp viruses, along with WSSV and YHV. TSV is a single-stranded RNA virus. ④ MrNV, the virus that causes white-tail disease (WTD), is a single-stranded RNA of Macrobrachium rosenbergii. It consists of two segments, RNA-1 and RNA-2. MrNV can be transmitted horizontally and vertically. Vertical transmission of infected shrimp is the main cause of shrimp fry disease. It can also be transmitted horizontally through virus-carrying water, biological baits such as rotifers, tools, and fish fry ponds that are not thoroughly disinfected. ⑤ XSV, a satellite virus related to MrNV, is a single-stranded positive RNA virus that was first discovered in Thailand and has been prevalent in many countries and regions. When XSV and MrNV are co-infected, they will cause a large number of Macrobrachium rosenbergii seedlings to die in a short period of time, and the muscles of diseased shrimps will be turbid. ⑥ CMD caused by Nodamura virus CMNV is a single-stranded RNA virus. The diseased shrimp mainly die in the deep water area at the bottom of the pond, which is not easy to be observed, so it is called CMD or bottom death disease. Diseased individuals show symptoms such as lighter color and atrophy of the hepatopancreas, empty jejunum and stomach, slow growth, and often opaque or partially white muscles of the abdominal segments of diseased shrimp. The mortality rate of diseased shrimp increases when the water temperature is high (above 28°C), and the cumulative mortality rate can reach 80%.

[0004] Pathogenic bacteria include the virulence factors PirA / B that cause acute hepatopancreatic necrosis disease (AHPND) in shrimp, which is characterized by sudden, mass mortality (mortality up to 100%) within a short period of time (usually within 30-35 days of stocking postlarvae or juvenile shrimp into grow-out ponds). Larger juvenile shrimp may also be affected. In areas where AHPND is endemic in shrimp farms, evidence suggests that the infection rate is close to 100%. Signs of disease in dying shrimp include a lighter to white coloration of the liver and pancreas due to loss of pigment in the connective tissues, and behavioral changes may also be observed, such as frequent sinking to the bottom of the water. In addition, there are Vibrio parahaemolyticus and Vibrio harveyi, Vibrio marinum, which causes vibriosis, Streptococcus indica, which can cause streptococcal disease, and Pseudomonas fluorescens, which can cause infection and mortality.

[0005] Enterocytosis of shrimp (EHP) is an obligate intracellular parasite. After infection with EHP, shrimp growth is retarded and economic losses are serious. It is a new and important pathogen that has endangered the development of the shrimp industry in recent years.

[0006] Shrimp pathogen detection often requires rapid, efficient, comprehensive and accurate detection, but the currently used nucleic acid detection methods cannot meet these requirements. Various molecular detection methods still have great limitations. The national standard methods and industry standards for the detection of various pathogens mostly use PCR or nested PCR or fluorescent quantitative PCR. However, the minimum detection limit of the PCR method is low, the steps of the nested PCR method are relatively cumbersome, the detection index is single, and it is easy to produce nucleic acid contamination. The fluorescent quantitative PCR method uses a dye method to detect pathogens, and it is impossible to control the quality of the extraction process. In addition, the melting curve method is easy to cause misinterpretation when there are many indicators. The detection of the fluorescent quantitative PCR TagMan probe method requires a standard curve for quantification, and has high requirements for the amplification efficiency of the primer probe. For most four-channel fluorescent quantitative PCR instruments, it is impossible to detect more than 4 pathogens in a single tube.

[0007] Establishing a rapid, efficient, comprehensive, accurate, highly sensitive and highly specific method for detecting multiple pathogens is not only crucial for the diagnosis and prevention of shrimp diseases, but also plays a guiding role in treatment and safe drug management. Efficient, rapid and accurate detection of pathogenic microorganisms in farmed shrimp can improve farming efficiency and reduce economic losses, and has great application value. Summary of the invention

[0008] The purpose of the present application is to provide a super-multiplex PCR primer pair, probe, microfluidic cartridge, kit, detection system and detection method for specific detection of shrimp pathogen microorganisms and parasites, which can quickly, accurately and ultra-high sensitivity detect 18 pathogens and virulence factors that cause farmed shrimp diseases. The PCR primer pair, probe, microfluidic cartridge, kit, detection system and detection method can be used for any PCR detection system, including but not limited to conventional PCR, RL-PCR, RNA-PCR, fluorescent quantitative PCR, digital PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELISA), nested PCR-high resolution melting analysis (nPCR-HRM), etc., preferably for digital PCR. For example, the purpose of the present application is to provide a PCR primer pair, probe, microfluidic cartridge, kit, detection system and detection method for specific detection sequences.

[0009] In order to achieve the above objectives, this application adopts the following technical solutions:

[0010] The present application relates to a combination for specifically detecting target genes, wherein the combination includes 18 pairs of primers for amplifying 18 target genes and 18 probes for hybridizing the 18 genes, 5 probes are respectively labeled with 3 concentrations of 2 different fluorescent groups, and the type or concentration of the fluorescent group carried by each of the 5 probes is different; at the same time, 1 probe is labeled with 1 concentration of 1 fluorescent group, so that 11 target genes can be detected simultaneously in 1 hole of the chip using 5 fluorescences.

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

[0012] The first group of primers and probes are: a PCR primer and probe combination for specifically amplifying a specific fragment of virulence factor PirA / B, the primers comprising an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2, and the probe comprising a probe as shown in SEQ ID NO:3; a PCR primer and probe combination for specifically amplifying a specific fragment of WSSV virus, the primers comprising an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5, and the probe comprising a probe as shown in SEQ ID NO:6; a PCR primer and probe combination for specifically amplifying a specific fragment of IMNV virus, the primers comprising an upstream primer as shown in SEQ ID NO:7 and a downstream primer as shown in SEQ ID NO:8, and the probe comprising 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, the primers comprising an upstream primer as shown in SEQ ID NO:10 and a downstream primer as shown in SEQ ID NO:11, and the probe comprising a probe as shown in SEQ ID NO:12. NO:12; a PCR primer and probe combination for specifically amplifying a specific fragment of TSV virus, the primer comprising an upstream primer as shown in SEQ ID NO:13 and a downstream primer as shown in SEQ ID NO:14, and the probe comprising a probe as shown in SEQ ID NO:15; a PCR primer and probe combination for specifically amplifying a specific fragment of IHHNV virus, the primer comprising an upstream primer as shown in SEQ ID NO:16 and a downstream primer as shown in SEQ ID NO:17, and the probe comprising a probe as shown in SEQ ID NO:18; a PCR primer and probe combination for specifically amplifying a specific fragment of shrimp hepatocellular carcinoma EHP, the primer comprising an upstream primer as shown in SEQ ID NO:19 and a downstream primer as shown in SEQ ID NO:20, and the probe comprising 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, the primer comprising an upstream primer as shown in SEQ ID NO:22 and a downstream primer as shown in SEQ ID NO:23, and the probe comprising a probe as shown in SEQ ID NO:24;

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

[0014] In a specific embodiment, the target gene is a specific fragment of pathogenic microorganisms and parasites that cause various shrimp diseases.

[0015] In a specific embodiment, the pathogen fragment is 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 harveyi VA, Streptococcus SI, and Pseudomonas fluorescens PF.

[0016] In a specific embodiment, 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 in the same reaction system, the spectral ranges of different types of fluorescent reporter groups are different.

[0017] In a specific 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 for specifically detecting 18 pathogens or virulence factor sequences of the present application is a combination used in digital PCR (dPCR) reaction.

[0019] The present application also relates to a digital PCR reaction premix for specifically detecting pathogens or virulence factor sequences, wherein the digital PCR reaction premix comprises the above-mentioned combination of the present application for specifically detecting 18 pathogens or virulence factor sequences.

[0020] In a specific embodiment, the digital PCR reaction premix 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, NH4 +, and Mg 2+ ion.

[0021] The present application also relates to a digital PCR microfluidic cartridge, wherein the digital PCR microfluidic cartridge comprises the digital PCR reaction premix for specifically detecting pathogens or virulence factor sequences of the present application.

[0022] In a specific embodiment, the microfluidic cartridge comprises at least one hole or at least two holes.

[0023] The present application also relates to a kit for detecting a target gene, wherein the kit includes the digital PCR reaction premix for specifically detecting pathogens or virulence factor sequences of the present application.

[0024] In a specific embodiment, the kit further comprises a positive quality control product, a negative quality control product, and / or a droplet generating oil.

[0025] In a specific embodiment, the kit further comprises the digital PCR microfluidic cartridge of the present application.

[0026] The present 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 nucleic acid from the sample to be tested;

[0028] (2) preparing a digital PCR amplification mixture, which contains the nucleic acid of the sample to be tested provided in step (1), the combination for specifically detecting 18 pathogens or virulence factor sequences of the present application, and / or the digital PCR reaction premix for specifically detecting pathogens or virulence factor sequences of the present application;

[0029] (3) preparing droplets in a digital PCR microfluidic cartridge and performing a PCR amplification reaction;

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

[0031] In the method for detecting target genes for non-disease diagnosis purposes described in the present application, the sample to be tested is selected from water, soil, shrimp feed and / or shrimp tissue in a shrimp farming base.

[0032] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features described in detail below can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described in detail.

[0033] The beneficial effect achieved by the present application is to provide a primer probe set for detecting a variety of shrimp pathogenic pathogens or virulence factor sequences, wherein the 5' end of the probe sequence of the present application is labeled with a fluorescent group of different colors and different concentrations, and the target sequence of each target gene is efficiently amplified by digital PCR, so as to achieve absolute quantification of each target gene to be detected, and directly give the number of copies per microliter (CPS / μl) of each target gene in the reaction system. In addition, the present application also provides a method for detecting target pathogens or virulence factors for the purpose of non-disease diagnosis using the primer and probe set of the method of the present application. For example, the combination of the present application can be used to detect a variety of pathogens or virulence factors in artificially cultured shrimp tissues. The sensitivity and specificity of the method of the present application are 95% and 100%, respectively, which exceed the prior art; and the minimum detection limit is 500 cps / ml, which is much lower than the minimum detection limit of the existing qPCR technology for directly detecting diseased shrimp tissue samples.

[0034] In particular, 1) a digital PCR detection kit is used to directly detect nucleic acids of pathogens or virulence factors in 200 μL shrimp tissues, and there is no need for a separate reverse transcription step for RNA viruses. DNA viruses, RNA viruses, bacteria, parasites and virulence factors can be detected simultaneously in one step; 2) a digital PCR primer probe set fluorescence mixed color coding technology is used to achieve single-hole detection of 11 pathogens; 3) detection targets include but are not limited to shrimp pathogens or virulence factors; 4) the detection kit contains an internal reference gene to prevent false negatives caused by insufficient sample extraction quality or PCR amplification reaction failure.

[0035] In a specific embodiment, the sample types involved in the detection include shrimp tissue, shrimp feed, environmental soil, and aquaculture water. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0037] Figure 1A-1F This is the positive control test result of simultaneously testing 18 pathogens and virulence factors plus 1 internal reference gene in 2 wells using the primer probe set of the present application in Example 1. Figure 1A , 1B and 1C are the test results of 7 pathogenic microorganisms and virulence factors detected in well 1; Figure 1D , 1E and 1F are the test results of the 11 pathogens detected in well 2.

[0038] Figure 2A-2C In order to optimize the detection efficiency of each shrimp pathogen by designing different primers and probes, the optimal primers and probes were selected for detection. Figure 2AThe results are shown in Table 1. The same concentration and volume of PirA / B positive plasmid was used for amplification using four sets of PirA / B primers and probes. Figure 2B The results are shown in Table 1. The same concentration and volume of WSSV-positive plasmid was used for amplification using four sets of WSSV primers and probes. Figure 2C The results are shown in Table 1. The same concentration and volume of IMNV-positive plasmid was used for amplification using four sets of IMNV primers and probes.

[0039] Figure 3 This is the specificity test result. The 18 pathogens and virulence factor detection kits reacted with 115 strains, and two wells were repeated. No positive droplets were produced in either well. This proves that the 18 pathogens detection kits have no non-specific cross-reactions with the 115 strains.

[0040] Figure 4A-4C The test results of shrimp tissue samples carrying pathogenic viruses or bacteria using the kit of the present application are shown. Figure 4A It indicates that WSSV-specific fragments were detected from shrimp carrying WSSV virus, and the droplets marked in orange are WSSV-positive droplets; Figure 4B It indicates that VP bacteria-specific fragments were detected from a shrimp tissue carrying Vibrio parahaemolyticus VP, and the blue-marked droplets are VP-positive droplets; Figure 4C It indicates that PirA / B virulence factor-specific fragments were detected from shrimp tissues carrying PirA / B virulence factors, and the blue-marked droplets are PirA / B-positive droplets.

[0041] Figure 5 This is the result of detecting a WSSV-positive sample of shrimp feed using the kit of the present application, wherein the droplets marked in orange are WSSV-positive droplets. DETAILED DESCRIPTION

[0042] The specific embodiments of the present application are described in detail below, but it should be understood that these embodiments are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following embodiments without specifying specific experimental conditions are usually operated under conventional conditions or under conditions recommended by the manufacturer.

[0043] Unless explicitly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising" etc. will be understood to include the stated elements or components but not to exclude other elements or components.

[0044] Digital PCR (dPCR) is a new absolute quantitative PCR technology that mainly involves limiting dilution of PCR reactants, followed by PCR amplification in different reaction chambers, and finally deriving the starting copy number or concentration of the target molecule based on the Poisson distribution principle and the number and ratio of positive droplets. Since there are multiple primers and probes in the multiplex digital PCR reaction system, the requirements for the primer-probe combination in the detection system are very high.

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

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

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

[0048] N is 1, 2 or 3, for example.

[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] Among them, the ratio range of any two fluorescent reporter groups A and B is 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 of any two fluorescent reporter groups A and B is in the range of 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, 1:6;

[0052] In the present application, A and B represent any fluorescent reporter groups, 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, and 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 the present application, when M is 4, N is 2, and X is 10, the identified pathogen P is 10. In one embodiment of the present application, when M is 5, N is 1, and X is 2, the identified pathogen P is 8.

[0055] In a specific manner, P probes are labeled respectively with N concentrations of M different fluorescent groups, and the type or concentration of the fluorescent group carried by each of the P probes is different, which means that the type of fluorescent group carried by each of the P probes is different, or the concentration of the fluorescent group carried by each of the P probes is different, or the type or concentration of the fluorescent group carried by each of the P probes is different. Those skilled in the art can understand that the core of this application is to use different types of fluorescent groups or fluorescence intensities to distinguish each probe, as long as they can be distinguished and can be detected by detection means.

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

[0057] In some embodiments, the gene of interest can be any type of shrimp disease-causing pathogen, including but not limited to the pathogens listed.

[0058] In some embodiments, the sequences of interest are multiple different shrimp pathogen sequences.

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

[0060] The combination of the present application can effectively control the number of multiple probes and utilize multiple different concentrations of multiple probes to specifically and efficiently detect multiple shrimp pathogens.

[0061] In one embodiment of the present application, the above-mentioned primer probe set is used to achieve simultaneous detection of 11 pathogens by 5-color fluorescence in 1 well and simultaneous detection of 18 pathogens and internal references by 5-color fluorescence in 2 wells through digital PCR. The detection range covers the most common viral, bacterial and parasitic pathogens in shrimp artificial breeding.

[0062] The primers and probes of the present application can be applied to any PCR detection system for detecting target genes, including but not limited to conventional PCR, RL-PCR, RNA-PCR, fluorescent quantitative PCR, digital PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELISA), nested PCR-high resolution melting analysis (nPCR-HRM), etc. It is preferred to use the primers and probes involved in the present application for fluorescent quantitative PCR or digital PCR. It is most preferred to use the primers and probes determined in the present application for digital PCR.

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

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

[0065] In some embodiments, the present application provides a digital PCR reaction premix for detecting a target gene, wherein the PCR reaction premix comprises the above-mentioned primer pairs, and a combination of each set of primer pairs and probes for any pathogen.

[0066] In a specific embodiment, the present application targets the specific nucleotide sequences of 18 shrimp pathogens and virulence factors, including 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, multiple digital PCR primers and probes that can be used to detect the above pathogens and virulence factors are designed, and up to 11-12 multiple digital PCRs can be detected in one well by using different concentrations of the probes. At the same time, internal standard primers and internal standard probes are also designed to monitor the sample collection and extraction process to avoid false negative results.

[0067] In order to improve the efficiency of screening shrimp pathogens and virulence factors, on the one hand, the present application provides a method for efficiently screening shrimp pathogens and virulence factors using a PCR method, wherein an example of an upstream primer for PirA / B is shown in SEQ ID NO: 1, and an example of a downstream primer is shown in SEQ ID NO: 2; an example of an upstream primer for WSSV is shown in SEQ ID NO: 4, and an example of a downstream primer is shown in SEQ ID NO: 5; an example of an upstream primer for IMNV is shown in SEQ ID NO: 7, and an example of a downstream primer is shown in SEQ ID NO: 8; an example of an upstream primer for VP is shown in SEQ ID NO: 10, and an example of a downstream primer is shown in SEQ ID NO: 11; an example of an upstream primer for TSV is shown in SEQ ID NO: 13, and an example of a downstream primer is shown in SEQ ID NO: 14; an example of an upstream primer for IHHNV is shown in SEQ ID NO: 16, and an example of a downstream primer is shown in SEQ ID NO: 17; an example of an upstream primer for EHP is shown in SEQ ID NO: 18. 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: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, the present application provides a probe that can screen the PirA / B gene, an example of which is shown in SEQ ID NO:3; a probe that can screen the WSSV gene, an example of which is shown in SEQ ID NO:6; a probe that can screen the IMNV gene, an example of which is shown in SEQ ID NO:9; a probe that can screen the VP gene, an example of which is shown in SEQ ID NO:12; a probe that can screen the TSV gene, an example of which is shown in SEQ ID NO:15; a probe that can screen the IHHNV gene, an example of which is shown in SEQ ID NO:18; a probe that can screen the EHP gene, an example of which is shown in SEQ ID NO:21; a probe that can screen the GAPDH gene, an example of which is shown in SEQ ID NO:24; a probe that can screen the YHV gene, an example of which is shown in SEQ ID NO:27; a probe that can screen the MBV gene, an example of which is shown in SEQ ID NO:30; a probe that can screen the SHIV gene, an example of which is shown in SEQ ID NO: NO:33; a probe that can screen HPV genes, an example of which is shown in SEQ ID NO:36; a probe that can screen VH genes, an example of which is shown in SEQ ID NO:39; a probe that can screen MrNV genes, an example of which is shown in SEQ ID NO:42; a probe that can screen XSV genes, an example of which is shown in SEQ ID NO:45; a probe that can screen CMNV genes, an example of which is shown in SEQ ID NO:48; a probe that can screen VA genes, an example of which is shown in SEQ ID NO:51; a probe that can screen SI genes, an example of which is shown in SEQ ID NO:54; a probe that can screen PF genes, an example of which is shown in SEQ ID NO:57.

[0069] In one aspect, the present application provides a combination of primers and probes that can simultaneously screen 18 pathogenic microorganisms and virulence factors, the combination of primers and probes comprising 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; SEQ ID NO: 31 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 the present application is labeled with a fluorescent group, and the 3' end of the probe sequence used in the present application is labeled with a fluorescent quenching group. When the above probes are located in the same reaction system, the spectral ranges of the fluorescent groups for screening different types of shrimp pathogens are different. The fluorescent group of the probe sequence used in the present 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 fluorescent quenching group of the probe sequence used in the present application can be any fluorescent quenching 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 the present application and the fluorescent reporter group labeled at the 5' end and the fluorescent quencher group labeled at the 3' end are as follows: the probe for screening PirA / B is shown in SEQ ID NO:3, whose 5' end is fluorescently labeled with FAM and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-CATTTATGGCTGGCGGCTGGAAAG-3'-BHQ1; the probe for screening WSSV is shown in SEQ ID NO:6, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TGGTATCCTCTTTCGCATTCGCCC-BHQ1-3', 5'-HEX-TGGTATCCTCTTTCGCATTCGCCC-BHQ1-3'; the probe for screening IMNV is shown in SEQ ID NO:9: its 5' end is fluorescently labeled with HEX, and its 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-TACAATGTCGGAGCGCAGGAACAG-BHQ1-3'; the probe for screening VP is shown in SEQ ID NO:12, its 5' end is fluorescently labeled with ROX, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TGGCGAACGAGAACGCAGACATTA-BHQ2-3'; the probe for screening TSV, an example of which is shown in SEQ ID NO:15, its 5' end is fluorescently labeled with ROX / Cy5, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TCCTCACACAACAACGCATAA-BHQ2-3', 5'-Cy5-TCCTCACACAACAACGCATAA-BHQ2-3'; the probe for screening IHHNV is shown in SEQ ID NO:18, whose 5' end is fluorescently labeled with ROX / Cy5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TGGTTTGTCCCACTTCATTTGGCC-BHQ2-3', 5'-Cy5-TGGTTTGTCCCACTTCATTTGGCC-BHQ2-3'; the probe for screening EHP is shown in SEQ ID NO:21, whose 5' end is fluorescently labeled with Cy5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-Cy5-TGCATGGCCGTTGGAAATTGATGG-BHQ2-3'; the probe for screening GAPDH gene is shown in SEQ ID NO:24: whose 5' end is fluorescently labeled with Cy5.5, and whose 3' end is fluorescently labeled with BHQ3, i.e., 5'-Cy5.5-TGTGTTCACCACCATCGAGAAGGC -BHQ3-3';The probe for screening YHV is shown in SEQ ID NO: 27, whose 5' end is fluorescently labeled with FAM and whose 3' end is fluorescently labeled with BHQ1, 5'-FAM-CGTCTCACACACATGGACTTCACCT-BHQ1-3'; the probe for screening MBV is shown in SEQ ID NO: 30, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TGCTGGGAATTTGCTAAAGTAACGACG-BHQ1-3', 5'-HEX-TGCTGGGAATTTGCAAAGTAACGACG-BHQ1-3'; the probe for screening SHIV is shown in SEQ ID NO:33: its 5' end is fluorescently labeled with FAM / HEX, and its 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-ACGTTAAAGGGTCTCACGGGAAACG-BHQ1-3', 5'-HEX-ACGTTAAAGGGTCTCACGGGAAACG-BHQ1-3'; the probe for screening HPV is shown in SEQ ID NO:36: its 5' end is fluorescently labeled with FAM / HEX, and its 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-ACTGTTCATCGTATCTTCCGCCGC-BHQ1-3', 5'-HEX-ACTGTTCATCGTATCTTCCGCCGC-BHQ1-3'; the probe for screening VH is shown in SEQ ID The probe for screening MrNV is shown in SEQ ID NO:39, whose 5' end is fluorescently labeled with HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-CGCATAATACCTTCGGGTCAAAGAGGG-BHQ1-3'; the probe for screening MrNV is shown in SEQ ID NO:42, whose 5' end is fluorescently labeled with ROX and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-CCTGGTAGTTCCCGAAGCGAATGT-BHQ2-3'; the probe for screening XSV is shown in SEQ ID NO:45, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-CCATGATCCTCGCATCGTATCGCA-BHQ2-3', 5'-Cy5-CCATGATCCTCGCATCGTATCGCA-BHQ2-3'; the probe for screening CMNV is shown in SEQ ID NO: As shown in NO:48, its 5' end is fluorescently labeled with ROX / Cy5, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TTGTATGGTGGAGCTGCGTCAAGT-3', 5'-Cy5-TTGTATGGTGGAGCTGCGTCAAGT-BHQ2-3';The probe for screening VA is shown in SEQ ID NO:51, and its 5' end is fluorescently labeled with ROX / Cy5, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-ACACAACAACCATTGTGGGTGGC-BHQ2-3', 5'-Cy5-ACACAACAACCATTGTGGGTGGC-BHQ2-3'; the probe for screening SI gene is shown in SEQ ID NO:54, and its 5' end is fluorescently labeled with Cy5, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-Cy5-TGCAGTTAAAGGGTCCGTAGTCGT-BHQ2-3'; the probe for screening PF gene is shown in SEQ ID NO:57, and its 5' end is fluorescently labeled with Cy5.5, and its 3' end is fluorescently labeled with BHQ3, i.e., 5'Cy5.5-ATCCACTTCAGCTGGGACATCCTG-BHQ3-3'. ;

[0072] In some embodiments, the digital PCR reaction premix provided in the present application, in addition to the above primer pairs, or the combination of any of the above primer pairs and probes, may 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, NH4 + and / or Mg 2+ ion.

[0073] The present application provides a kit for detecting a target gene, which includes the primer pair mentioned above, or a combination of any primer pair and a probe mentioned above, or a combination of the primer pair and any probe mentioned above. The kit of the present 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 kit of the present application may also include a positive control and / or a negative control.

[0076] In some embodiments, the present 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] The sample to be tested is 100-1000 μL, preferably 100-200μl, 600-1000μl, 700-1000μl, 800-1000μl, 900-1000μl, and most preferably 1000μl to increase the detection rate. Centrifuge at 800 rpm for 10 minutes to take the supernatant. The separated sample can be stored at -20℃±5℃ for 3 years. It is recommended to use a commercial kit to extract total nucleic acid including DNA and RNA in the sample to be tested. After the nucleic acid extraction is completed, it is recommended to test immediately, otherwise it is best to store it below -20℃ for no more than 3 months.

[0079] (2) Prepare a digital PCR amplification mixture according to Table 1, which contains the sample to be detected provided in step (1) and the following digital PCR reaction premix; 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; the final concentrations of each probe are 50-800 nM, preferably 50-500 nM, 100-400 nM, and most preferably 250 nM; Mg 2+ The final concentration of 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 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 / reaction, 1.5-4 U / reaction, 2-3.5 U / reaction, and optimally 2.5 U / reaction; the final concentration of reverse transcriptase is 0.5-10 U / reaction, preferably 1-5 U / reaction, 1.5-4 U / reaction, 2-3.5 U / reaction, and optimally 2.5 U / reaction; the final concentration of dNTP is 20-300µM, preferably 50-250µM, 100-230µM, 150-220µM, and most preferably 200µM. The digital PCR reaction premix may also contain UNG enzyme, and its final concentration is 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 value of the digital PCR reaction premix is ​​7-9, preferably pH7.4-8.5, preferably pH 7.8-8.2, and most preferably pH 8.0.

[0080] Those skilled in the art will appreciate that the substances and concentrations listed in the above PCR amplification mixture are illustrative only, and those skilled in the art can make selections based on the actual system.

[0081] Table 1. Digital PCR amplification mixture

[0082]

[0083] (3) preparing droplets and performing PCR amplification reaction;

[0084] Step 1: Prepare microdroplets. First, add 50-100 μl of droplet generation oil into the square filling hole on 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, primer probe, etc.) into the circular sample well on the top of the microfluidic cartridge, preferably 15-25 μl, most preferably 20 μl;

[0086] Step 3: Cover the sample loading hole and oil filling hole at the top of the microfluidic cartridge and the waste liquid hole at the bottom with two sealing covers respectively; 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] Select DQ mode on the biochip reader (e.g., Dscanner4-1000, registration number: Su Xie Zhu Zhun 20202220818), set FAM, HEX, ROX, Cy5 and Cy5.5 as the fluorescence channels, amplify the high-throughput digital PCR cartridge (e.g., microfluidic cartridge) from the PCR amplifier, preferably the SG-2000 PCR amplifier (registration number: Su Xie Zhu Zhun 20202220887), take it out after the reaction is completed, transfer it smoothly and place it on the biochip reader for signal collection.

[0091] (5) Results analysis

[0092] Click "Analyze" on the biochip reader to analyze the experimental data. First check that "Accepted Droplets" ≥ 10,000 before proceeding with the subsequent analysis. Click "2D Amplitude" to view the 2D scatter plots of channel TAM, channel HEX, channel ROX, and channel Cy5. According to the positive control, click on the 2D scatter plot to divide the positive droplets in each area. In the 2D scatter plot with FAM as the Y axis and HEX as the horizontal axis (for example Figure 1D ), the darkest black dots represent droplets without amplification templates, the lake blue dots represent droplets at position 1 containing FAM labeled probes, the green dots represent droplets at position 2 containing both FAM / HEX labeled probes, the orange dots represent droplets at position 3 containing both FAM / HEX labeled probes, the purple dots represent droplets at position 4 containing both FAM / HEX probes, and the brown-gray dots represent droplets at position 5 containing HEX probes. In a 2D scatter plot with ROX as the Y axis and Cy5 as the horizontal axis (e.g. Figure 1E ), the darkest black dots represent droplets without amplified templates, the lake blue dots represent droplets at position 1 containing ROC labeled probes, the green dots represent droplets at position 2 containing both ROX / Cy5 labeled probes, the orange dots represent droplets at position 3 containing both ROX / Cy5 labeled probes, the purple dots represent droplets at position 4 containing both ROX / Cy5 probes, and the brown-grey dots represent droplets at position 5 containing Cy5 probes. 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, and can also be used alone or together with other reagents for the detection of multiple pathogens in shrimp tissues, wherein the processing steps for shrimp tissue samples are: 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, 900-1000 μl, and most preferably 1000 μl to improve the detection rate. To improve the detection rate. Use tissue grinding fluid to make the shrimp tissue into a pulp. Take 200 μl of shrimp pulp for nucleic acid extraction. It is recommended to use a commercial kit to extract microbial nucleic acids from shrimp pulp samples. After the nucleic acid extraction is completed, it is recommended to test immediately, otherwise it is best to store it below -20°C for no more than 3 months.

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

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

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

[0097] Table 4. Examples of fluorescent labels for probes used in this application

[0098]

[0099] Example

[0100] Example 1. Kit for detecting multiple pathogens in shrimp pathogen infection

[0101] The present application develops a digital PCR detection kit for detecting 18 pathogens and virulence factors plus 1 internal reference gene in shrimp tissues. The above 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 quality control and positive quality control (see Table 7 for specific examples of positive quality control and negative quality control).

[0102] Each sample needs to undergo three sets of tests. The primer-probe mixture and digital PCR premix in the kit are used to generate digital PCR droplets, perform reverse transcription, and perform PCR amplification on the sample to be tested. The volume of the primer-probe mixture is 2 μL, the volume of the digital PCR premix 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:

[0103] Table 5: Group 1 Multiplex Digital Reaction System

[0104]

[0105] Table 6: Group 2 Multiplex Digital Reaction System

[0106]

[0107] The components of the kit of the present application are shown in Table 7.

[0108] Table 7: Composition of the kit

[0109]

[0110] An internal quality control system is set up in the kit of this application to monitor the sample collection and extraction process to avoid false negatives. Negative quality control products and positive quality control products are set up in the kit of this application (see Table 7 for specific examples of positive quality control products and negative quality control products) to monitor the digital PCR amplification and chip reading of the sample, and to assist in the interpretation of the final results. The specific steps are as follows:

[0111] 1) Release nucleic acid from the sample to be tested;

[0112] 2) In one well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, 7 pathogens, virulence factor (PirA / B) and internal reference (GAPDH) primers and a fluorescent labeled probe mixture (corresponding to group 1, see Table 7) and digital PCR buffer were added;

[0113] 3) In another well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, another 11 pathogen primers and fluorescent labeled probe mixtures (corresponding to group 2, see Table 7) and digital PCR buffer were added;

[0114] 4) Perform digital PCR droplet generation, reverse transcription, and PCR amplification in a digital PCR amplifier; add 75 μl of oil-phase encapsulated reagent to the oil wells in the high-throughput digital PCR chip, add 20 μl of amplification mixture to the reagent wells, cover with a sealing cover, and place on a PCR amplifier for droplet generation, reverse transcription, and PCR amplification.

[0115] Perform reverse transcription and PCR amplification reactions according to the following recommended amplification protocols:

[0116] Table 8. Reverse transcription and PCR amplification procedures

[0117]

[0118] 5) Signal collection

[0119] The nucleic acid obtained in step 4) is used as a template, and the chip after amplification is used as a test object. The fluorescence is detected by a biochip reader, and the results are interpreted by the presence and intensity of fluorescence in each droplet.

[0120] Result interpretation:

[0121] The results are shown in Figure 1 and Table 9, with 7 pathogenic microorganisms and virulence factors detected in the first well and 11 pathogens detected in the second well.

[0122] Table 9. Pathogens and virulence factors corresponding to different positions of wells 1 and 2

[0123]

[0124] If in the 2D scatter plot, in addition to negative droplets, there are positive droplets in the positive droplet area, and the number of droplets is greater than the threshold, the strain detection result corresponding to the position is determined to be positive.

[0125] Table 10. Positive groups corresponding to shrimp pathogens and virulence factors

[0126]

[0127] Example 2. Optimizing the detection efficiency of different primers and probes for pathogens

[0128] For each shrimp pathogenic microorganism, 2-4 sets of primers and probes were designed for detection. The primer and probe pairs used in the experiment are shown in Table 3 and Table 12. For each pathogen, the primers and probes in Table 3 and Table 12 were used to prepare primer-probe mixture (mix 2-4 sets of primers and probes), digital PCR premix (digital PCR buffer, dNTPs, MgCl2, taq hot start enzyme mixture, reverse transcriptase, glycerol, betaine), and the positive quality control products for the pathogens targeted by the corresponding primers and probes (see Table 7 for specific examples of positive quality control products and negative quality control products).

[0129] Each set of primer probes for each pathogen needs to be tested in 2 groups, and the primer probe mixture and digital PCR premix in the kit are used to generate digital PCR droplets, reverse transcribe and amplify the sample to be tested. The volume of the primer probe mixture is 2 μL, the volume of the digital PCR premix 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 rest of the operations are the same as in Example 1.

[0130] Table 11: Digital reaction system for primer and probe optimization

[0131]

[0132] Set 1 refers to the primers and probes described in Table 3, and Sets 2 to 4 refer to the primers and probes described in Table 12.

[0133] Table 12. Primer and probe sequences used in the optimization of 18 microbial pathogens, virulence factors and internal reference genes in this application ; .

[0134] 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. Figure 2A-2C The test results for three of these pathogens are listed in the table. Figure 2AThe results are shown in Table 3. PirA / B-1 (i.e., PirA / B-f1, PirA / B-r1, and PirA / B-P1 described in Table 3) was detected with the largest number of copies, and this set of primers and probes was selected for use in the kit. Figure 2B The results of amplification using 4 sets of WSSV primers and probes with the same concentration and volume of WSSV positive plasmid were shown. The copy number of WSSV-1 (i.e., WSSV-f1, WSSV-r1, and WSSV-P1 described in Table 3) was the highest, and this set of primers and probes was selected and put into the kit for use. Figure 2C The results are shown in Table 3. The same concentration and volume of IMNV positive plasmid was used to amplify the results using 4 sets of IMNV primers and probes. IMNV-1 (i.e., IMNV-f1, IMNV-r1, and IMNV-P1 described in Table 3) had the largest number of copies detected, and this set of primers and probes was selected and put into the kit for use.

[0135] Example 3. Specific detection

[0136] The present application selects purple bacillus, intermedius streptococcus, bronchiseptica Bordetella, marcescens serratia, sobrinus streptococcus, listeria ei, cepacia Burkholderia, anginosus streptococcus, anaerobic peptostreptococcus, sanguis streptococcus, constellation streptococcus constellation subspecies, Veillonella parvula, agalactiae streptococcus, harmless listeria, banded bacillus, sobria aeromonas, salivarius streptococcus, caviae aeromonas, terrestrial raoulella (terrestrial klebsiella), capitate staphylococcus capitate subspecies, dysgalactiae streptococcus, micrococcus luteus, sonnei shigella, squirrel staphylococcus, listeria monocytogenes, aquatic raenella, salmonella typhimurium, lactobacillus acidophilus, hydrophilic aeromonas Bacteria, Bacillus agglutinans, Legionella pneumophila subspecies, Staphylococcus stutzeri subspecies, Pseudomonas stutzeri, Enterobacter carcinogenes, Sphingomonas paucimobilis, Neisseria lactis, Staphylococcus carnosus, Japanese Gouweiduo bacteria, Streptococcus canis, Enterococcus lead yellow, Proteus mirabilis, Pseudomonas rice, Bacteroides vulgaris, Acinetobacter pitei, Ralstonia pitei, Streptococcus bovis, Eikenella corrodens, Streptococcus pyogenes, Neisseria meningitidis, Enterococcus durable, Staphylococcus xylosus, Moraxella morrhagica, Morganella morganii, Pseudomonas mendocinosae, Enterococcus caecum, Streptococcus equi zooepidemicus subspecies, Rhodococcus equi, Basiliella marneffei, Gemini cocci of measles, Bacteroides ovale, Staphylococcus lugdunensis, Acinetobacter lwoffii, Haemophilus influenzae, Neisseria gonorrhoeae, Shigella dysenteriae, Oleomonas shigellae, Bacillus cereus, Bacillus subtilis, Streptococcus oralis, Acinetobacter radioresistance, Enterobacter cobe, Kingella, Streptococcus gallolyticus, Staphylococcus pseudointermedia, Enterobacter hallii, Streptococcus mitis, Enterococcus hirae, Listeria monocytogenes, Streptococcus gordonii, Neisseria dry, Streptococcus parasanguinis, Clostridium difficile, Bordetella parapertussis, Staphylococcus saprophyticus, Shigella flexneri, Yersinia flexneri, Hafnia alvei, Photobacterium non-symbioticum, Staphylococcus auris, Pseudomonas putida, Bacteroides thetaiotaomicron, Burkholderia polyphaga Specific reactions were performed on 115 strains, including Delbrueckia spp., Pasteurella multocida, Parabacteroides dieldii, Bacillus licheniformis, Bacteroides monotheca, Listeria monocytogenes, Propionibacterium acnes, Vibrio vulnificus, Pantoea agglomerans, Salmonella enteritidis, Yersinia enterocolitica, Klebsiella oxytoca, Pseudomonas alcaligenes, Streptococcus mutans, Acinetobacter baylyi, Acinetobacter berreitzii, Shigella boydii, Actinobacillus actinomycetemcomitans, Cronobacter sakazakii, Bordetella pertussis, Enterococcus gallinaceus, Clostridium perfringens, Staphylococcus warrenii and Haemophilus parainfluenzae. These strains are common pathogens that are prone to cause clinical symptoms in clinical practice. It was confirmed that none of the above strains were detected.

[0137] (1) releasing nucleic acid of the strain to be tested;

[0138] (2) mixing the nucleic acids extracted from the above strains;

[0139] (3) Digital PCR droplet formation and amplification:

[0140] Digital PCR droplet formation and digital PCR amplification were performed with reference to the reaction system and reaction procedure described in Example 1.

[0141] (4) Signal collection:

[0142] The digital PCR chip was removed from the digital PCR amplification instrument, and was smoothly transferred to the biochip reader for signal collection.

[0143] (5) Result interpretation and analysis:

[0144] The sample is drawn with a threshold line at the middle position between the negative droplet and the positive droplet of the positive quality control in the kit. If the number of positive droplets in the mixed sample is greater than the threshold, the test result of the mixed strain of bacteria corresponding to the positive droplet is determined to be positive. The software gives the copy number corresponding to the positive droplet. A linear regression graph is drawn with the dilution as the horizontal axis and the measured value log of each dilution as the vertical axis.

[0145] The results of cross-reaction detection of 115 strains are as follows Figure 3 As shown, no cross-reactions were detected between these 115 strains and the 18 pathogen primer probes in the kit. Figure 3 Only negative droplets were present, and no positive droplets were detected that were cross-reactive with the 115 strains, indicating that the primers and probes described in the present application have good detection specificity.

[0146] Example 4. Detection kit for samples of bacterial strains carrying drug-resistant genes

[0147] The samples carrying shrimp pathogens were tested according to the method of Example 1. The diseased shrimp samples were from a shrimp pond in Nantong. Figure 4A Indicates the test result of WSSV virus, indicating that WSSV virus gene was detected in the shrimp tissue sample; Figure 4B The test results of VP indicate that VP bacteria were detected in the shrimp tissue samples tested; Figure 4C It indicates a positive result for the detection of PirA / B virulence genes, indicating that PirA / B virulence factors were detected in the sample.

[0148] Example 5. Detection of pathogens in shrimp feed samples using the detection kit

[0149] Referring to the method of Example 1, 14 samples of water, soil, shrimp feed and shrimp tissue obtained from a shrimp farming base in a certain place were tested.

[0150] The results showed that VSSV virus was detected in one sample ( Figure 5 ).

[0151] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A PCR primer and probe combination for specific detection of target genes of multiple pathogens in shrimp, the primers and probes comprising primers and probes for any one or more target genes of pathogens in the following two groups of primers and probes, wherein: The first set of primers and probes are: a PCR primer and probe combination for specifically amplifying a specific fragment of virulence factor PirA / B, wherein the primers include an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, and the probe includes 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 probe includes 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 probe includes 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 probe includes 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 probe includes a probe as shown in SEQ ID NO: 18; A PCR primer and probe combination for specifically amplifying a specific fragment of shrimp hepatocellular carcinoma 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 a probe as shown in SEQ ID NO: 21; A PCR primer and probe combination for specifically amplifying a specific fragment of an 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 are: a PCR primer and probe combination for specifically amplifying a specific fragment of the YHV virus, wherein the primers include an upstream primer as shown in SEQ ID NO: 25 and a downstream primer as shown in SEQ ID NO: 26, and the probe includes a probe as 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 an upstream primer as shown in SEQ ID NO:28 and a downstream primer as shown in SEQ ID NO:29, and the probe includes 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 probe includes a probe as shown in SEQ ID NO:33; A PCR primer and probe combination for specifically amplifying a specific fragment of HPV virus, the primers comprising 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; a PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio harveyi VH, the primers comprising 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 probe includes 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 probe includes 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 probe includes a probe as shown in SEQ ID NO:48; A PCR primer and probe combination for specifically amplifying a specific fragment of Vibrio algae 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 SI, wherein the primers include an upstream primer as shown in SEQ ID NO:52 and a downstream primer as shown in SEQ ID NO:53, and the probe includes a probe as shown in SEQ ID NO:54; A PCR primer and probe combination for specifically amplifying a specific fragment of Pseudomonas fluorescens PF, wherein the primers include an upstream primer as shown in SEQ ID NO:55 and a downstream primer as shown in SEQ ID NO:56, and the probe includes a probe as shown in SEQ ID NO:

57.

2. According to the PCR primer and probe combination of claim 1, 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 in the same reaction system, the spectral range of the fluorescent reporter group for screening different target genes is different.

3. The PCR primer and probe combination 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 PCR primer and probe combination according to claim 3, wherein each probe used and its 5' end labeled fluorescent reporter group and 3' end labeled fluorescent quencher group are as follows: The probe for screening PirA / B is shown in SEQ ID NO:3, and its 5' end is fluorescently labeled with FAM and its 3' end is fluorescently labeled with BHQ1; the probe for screening WSSV is shown in SEQ ID NO:6, and its 5' end is fluorescently labeled with FAM / HEX and its 3' end is fluorescently labeled with BHQ1; the probe for screening IMNV is shown in SEQ ID NO:9, and its 5' end is fluorescently labeled with HEX and its 3' end is fluorescently labeled with BHQ1; the probe for screening VP is shown in SEQ ID NO:12, and its 5' end is fluorescently labeled with ROX and its 3' end is fluorescently labeled with BHQ2; the probe for screening TSV is shown in SEQ ID NO:15, and its 5' end is fluorescently labeled with ROX / Cy5 and its 3' end is fluorescently labeled with BHQ2; the probe for screening IHHNV is shown in SEQ ID NO:18, and its 5' end is fluorescently labeled with ROX / Cy5 and its 3' end is fluorescently labeled with BHQ2; the probe for screening EHP is shown in SEQ ID NO: NO:21, whose 5' end is fluorescently labeled with Cy5 and 3' end is fluorescently labeled with BHQ2; the probe for screening the internal reference GAPDH is shown in SEQ ID NO:24, whose 5' end is fluorescently labeled with Cy5.5 and 3' end is fluorescently labeled with BHQ3; the probe for screening YHV is shown in SEQ ID NO:27, whose 5' end is fluorescently labeled with FAM and 3' end is fluorescently labeled with BHQ1; the probe for screening MBV is shown in SEQ ID NO:30, whose 5' end is fluorescently labeled with FAM / HEX and 3' end is fluorescently labeled with BHQ1; the probe for screening SHIV is shown in SEQ ID NO:33, whose 5' end is fluorescently labeled with FAM / HEX and 3' end is fluorescently labeled with BHQ1; the probe for screening HPV is shown in SEQ ID NO:36, whose 5' end is fluorescently labeled with FAM / HEX and 3' end is fluorescently labeled with BHQ1; the probe for screening VH is shown in SEQ ID NO: The probe for screening MrNV is shown in SEQ ID NO:39, whose 5' end is fluorescently labeled with HEX and whose 3' end is fluorescently labeled with BHQ1; the probe for screening XSV is shown in SEQ ID NO:45, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2; the probe for screening CMNV is shown in SEQ ID NO:48, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2; the probe for screening VA is shown in SEQ ID NO:51, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2; the probe for screening SI is shown in SEQ ID NO:54, whose 5' end is fluorescently labeled with Cy5 and whose 3' end is fluorescently labeled with BHQ2; the probe for screening PF is shown in SEQ ID NO: As shown in NO:57, its 5' end is fluorescently labeled with Cy5.5, and its 3' end is fluorescently labeled with BHQ3.

5. The combination according to any one of claims 1 to 4, which is a combination used in a digital PCR (dPCR) reaction.

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

7. The digital PCR reaction premix according to claim 6, wherein the digital PCR reaction premix 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, NH4 + , and Mg 2+ ion.

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

9. The microfluidic cartridge of claim 8, wherein the microfluidic cartridge comprises at least one hole or at least two holes.

10. A kit for detecting a target gene, wherein the kit comprises a digital PCR reaction premix for specifically detecting a target gene according to claim 6 or claim 7.

11. The kit according to claim 10, wherein the kit further comprises a positive control, a negative control, and / or droplet generation oil.

12. The kit according to claim 11, wherein the kit further comprises a digital PCR microfluidic cartridge according to claim 9.

13. A method for detecting a target gene for a purpose other than disease diagnosis, the method comprising the following steps: (1) Release nucleic acid from the sample to be tested; (2) preparing a digital PCR amplification mixture, which contains the nucleic acid of the sample to be detected provided in step (1), the combination according to any one of claims 1 to 5, and / or the digital PCR reaction premix according to claim 6 or 7; (3) preparing droplets in a digital PCR microfluidic cartridge and performing a PCR amplification reaction; (4) Use a digital PCR microfluidic card reader to read the fluorescence signal after the droplet PCR amplification reaction.

14. The method for detecting a target gene for non-disease diagnosis purposes according to claim 13, wherein the sample to be tested is selected from water, soil, shrimp feed and / or shrimp tissue of a shrimp farming base.

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