CN110387440B - Reagent for multiple detection of salmon and trout viruses and application thereof - Google Patents
Reagent for multiple detection of salmon and trout viruses and application thereof Download PDFInfo
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Abstract
The application discloses a reagent for multiple detection of salmon and trout viruses and application thereof. The reagent of the present application comprises a specific primer set comprising at least one of a first primer pair to a sixth primer pair; the first to sixth primer pairs are primer pairs for specifically amplifying spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order; the upstream and downstream primers of the first to sixth primer pairs are the sequences shown in Seq ID No.1 to Seq ID No.12, in that order. The reagent can carry out multiple detection on six salmon and trout viruses, is simple and convenient to use, shortens the detection time of the six salmon and trout viruses, improves the detection efficiency and quality, and has great significance for quick detection and prevention and control of the six salmon and trout viruses.
Description
Technical Field
The application relates to the field of salmon and trout virus detection, in particular to a reagent for multiple detection of salmon and trout viruses and application thereof.
Background
Salmon and trout are the collective term for Salmonidae (Salmonidae) fish, and include all species of the Salmonidae and Salmonidae families. The salmon and the trout enjoy the reputation of 'ginseng in water' because of tender meat, delicious taste and high nutritional value, and are high-quality breeding varieties popularized to all countries in the world by food and agriculture organizations in the united nations. The cultured salmon and trout are introduced from the last 70 th century in China, at present, the culture area of the salmon and trout reaches more than 1500 mu, the annual culture yield of the salmon and trout is about 3 ten thousand tons, and the method is one of the main cultured varieties of cold water fishes in China.
In recent years, salmon and trout epidemic diseases frequently occur, and huge economic losses are caused to farmers. It has been reported that viruses infecting fish such as salmon and trout mainly include Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alphavirus (SAV), Viral Hemorrhagic Septicemia Virus (VHSV), Spring Viraemia of Carp Virus (SVCV), and the like.
Viral diseases are highly contagious, often cause organ, tissue and cell damage resulting in temporary or permanent functional insufficiency or damage, regular model outbreaks, and high mortality. Infection with viral diseases can also lead to increased susceptibility of fish populations to other diseases with enormous collateral losses. Therefore, the monitoring of the diseases related to the salmon and the trout draws attention of relevant departments, but the detection cost is more and more important due to the increase of the types of the diseases.
At present, the detection methods of the viruses mainly comprise a tissue electron microscope, virus separation, RT-LAMP, ELISA, real-time fluorescence PCR and the like. The virus separation method has accurate detection result, but has long time consumption and high requirement on experimental conditions; the sensitivity and accuracy of the common PCR and real-time fluorescence PCR detection methods are greatly improved, but the detection method only has good effect on a single target at present and is easy to generate false positive results on the detection of multiple targets. The situation that various viruses are mixed to infect often appears in an actual sample, and the viruses are detected and analyzed one by adopting a common PCR or real-time fluorescence PCR detection method with a single target, so that the method is time-consuming and labor-consuming, and is not beneficial to rapid prevention and treatment of the viruses.
Therefore, the development of a method or means for rapidly and effectively screening and detecting the salmon and trout related diseases is urgently needed.
Disclosure of Invention
The application aims to provide a novel reagent for multiple detection of salmon and trout viruses and application thereof.
The following technical scheme is adopted in the application:
one aspect of the application discloses a reagent for multiple detection of salmon and trout viruses, which comprises a specific primer group, wherein the specific primer group comprises at least one pair of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair and a sixth primer pair; the first to sixth primer pairs are primer pairs for specifically amplifying spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order; the upstream and downstream primers of the first primer pair are respectively a sequence shown in Seq ID No.1 and a sequence shown in Seq ID No. 2; the upstream and downstream primers of the second primer pair are respectively a sequence shown in Seq ID No.3 and a sequence shown in Seq ID No. 4; the upstream primer and the downstream primer of the third primer pair are respectively a sequence shown in Seq ID No.5 and a sequence shown in Seq ID No. 6; the upstream primer and the downstream primer of the fourth primer pair are respectively a sequence shown in Seq ID No.7 and a sequence shown in Seq ID No. 8; the upstream primer and the downstream primer of the fifth primer pair are respectively a sequence shown in Seq ID No.9 and a sequence shown in Seq ID No. 10; the upstream primer and the downstream primer of the sixth primer pair are respectively a sequence shown in Seq ID No.11 and a sequence shown in Seq ID No. 12;
Seq ID No.1：5’-CAGGCCACGTATTGTCATGCTGCCAAATCACCATACTCAACAATAA-3’
Seq ID No.2：5’-TTCTTGGCGTTATGTCGCTGGAGTCAATCTCATCAGGCTGTCTTG-3’
Seq ID No.3：5’-CAGGCCACGTATTGTCATGCAGAGCCAAGGCACTGTGCG-3’
Seq ID No.4：5’-TTCTTGGCGTTATGTCGCTGTTCTTTGCGGCTTGGTTGA-3’
Seq ID No.5：5’-CAGGCCACGTATTGTCATGCAAACTCGCAGGATGTGTGCGTCC-3’
Seq ID No.6：5’-TTCTTGGCGTTATGTCGCTGTCTGCGATCTCAGTCAGGATGAA-3’
Seq ID No.7：5’-CAGGCCACGTATTGTCATGCCTACACAGCAGGATGCAGATGT-3’
Seq ID No.8：5’-TTCTTGGCGTTATGTCGCTGCAGGAGCCGGAAGTCGAT-3’
Seq ID No.9：5’-CAGGCCACGTATTGTCATGCCCGGCCCTGAACCAGTT-3’
Seq ID No.10：5’-TTCTTGGCGTTATGTCGCTGGTAGCCAAGTGGGAGAAAGCT-3’
Seq ID No.11：5’-CAGGCCACGTATTGTCATGCGAGGTCTCAGACTCCGGAAGTG-3’
Seq ID No.12：5’-TTCTTGGCGTTATGTCGCTGTCCAGCGCCGTCTGGTT-3’。
the reagent can realize multiple detection of spring viremia of carp virus, infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, infectious salmon anemia virus, salmon alphavirus and infectious pancreas necrosis virus, and six common and more harmful salmon and trout viruses through six pairs of specific primers, can quickly and effectively analyze and detect the six salmon and trout viruses at one time, shortens the detection time, improves the detection efficiency and quality, and provides a powerful scientific means for quickly detecting and preventing the six salmon and trout viruses.
Preferably, the reagent further comprises a universal primer pair, and the upstream primer and the downstream primer of the universal primer pair are respectively a sequence shown in Seq ID No.13 and a sequence shown in Seq ID No. 14;
Seq ID No.13：5’-CAGGCCACGTATTGTCATGC-3’
Seq ID No.14：5’-TTCTTGGCGTTATGTCGCTG-3’。
in the present application, when six pairs of specific primers are designed, a universal sequence unrelated to the specific detection target is added to the 5 ' ends of the upstream and downstream primers of the specific primer pair in advance, that is, a same universal sequence is added to the 5 ' ends of the upstream primers of all the specific primers, and another same universal sequence is added to the 5 ' ends of the downstream primers of all the specific primers. The universal primer pair has the advantages that the universal primer pair has the effect of simultaneously amplifying six different target fragments by adopting a pair of primers, so that the concentration and the content of the target fragments are effectively improved, and the subsequent detection is facilitated; therefore, the universal sequence added to the 5' ends of the upstream primer and the downstream primer of the universal primer pair or the specific primer pair may be any sequence that does not overlap with the target sequence or non-specifically amplify the target. The universal primer pair of the sequence shown in Seq ID No.13 and the sequence shown in Seq ID No.14 of the present application is only a universal primer pair which is proved to be applicable to multiplex detection in one implementation manner of the present application, and does not exclude that other universal sequences or universal primer pairs can also be adopted; if other universal primer pairs are adopted, the 5' terminal sequences of the corresponding six pairs of specific primers shown in Seq ID No.1 to Seq ID No.12 are also changed correspondingly, as long as the specific amplification of the six pairs of specific primers is not affected.
Preferably, in the universal primer pair, the 5' segment of the sequence primer shown in Seq ID No.14 has a biotin tag, and the fifth base and the thirteenth base in the sequence are both internally biotin-tagged deoxythymine.
Preferably, the reagent of the present application further comprises a specific probe set comprising at least one of the first probe, the second probe, the third probe, the fourth probe, the fifth probe and the sixth probe; the first to sixth probes are probes specifically hybridizing to spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order; the first probe is a sequence shown in Seq ID No.15, the second probe is a sequence shown in Seq ID No.16, the third probe is a sequence shown in Seq ID No.17, the fourth probe is a sequence shown in Seq ID No.18, the fifth probe is a sequence shown in Seq ID No.19, and the sixth probe is a sequence shown in Seq ID No. 20;
Seq ID No.15：5’-TTGTTGCTGCATTGTGCCGCTCC-3’
Seq ID No.16：5’-TGAGACTGAGCGGGACA-3’
Seq ID No.17：5’-TAGAGGGCCTTGGTGATCTTCTG-3’
Seq ID No.18：5’-CATCGTCGCTGCAGTTC-3’
Seq ID No.19：5’-CTGGCCACCACTTCGA-3’
Seq ID No.20：5’-CTTCTAGTCTGCTTCCCGGGTGCACC-3’。
it should be noted that six pairs of specific primers in the reagent of the present application can already realize multiplex detection of spring viremia of carp virus, infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, infectious salmon anemia virus, salmon alphavirus and infectious pancreatic necrosis virus, and six salmon and trout viruses, but in order to improve the accuracy, specificity and sensitivity of detection, six specific probes matched with the six pairs of specific primers are further developed, that is, the first to sixth probes are sequentially matched with the first to sixth primer pairs, i.e., the hybridization binding regions of the probes are located in the amplification range of the primer pairs. It can be understood that the six specific probes of the present application can be modified or processed according to the specific detection method, for example, if real-time fluorescence PCR detection is adopted, the 5 'ends of the six probes need to be labeled with different fluorescent groups, and the 3' ends are labeled with fluorescence quenching groups; if the gene chip is adopted for detection, six probes need to be fixed on the gene chip; if a liquid phase chip is adopted for detection, six probes are required to be fixed on the fluorescent coding microspheres with different numbers.
Preferably, in the reagent of the present application, the third base, the seventh base and the eighth base of the second probe having a sequence represented by Seq ID No.16 are all acid-locked modified bases.
Preferably, in the third probe having the sequence shown in Seq ID No.17, the seventh base and the eighth base are both acid-locked modified bases.
Preferably, in the fourth probe having the sequence represented by Seq ID No.18, the fifth base, the sixth base, the seventh base and the twelfth base are all acid-locked modified bases.
Preferably, in the fifth probe having the sequence represented by Seq ID No.19, the fifth base, the sixth base and the eighth base are all acid-locked modified bases.
Note that, a Locked acid modified base, i.e., a Locked Nucleic Acid (LNA) label, is used for the purpose of adjusting the dissolution temperature of a primer or a probe; the application creatively uses the probes in the application to ensure that six probes can reach relatively uniform dissolving temperature on the basis of keeping specificity so as to facilitate subsequent multiple detection.
Preferably, the 5' -end of each of the first to sixth probes has a carbon arm and an amination modification. Preferably, the carbon arm is a C6 arm or a C12 arm.
The application also discloses application of the reagent in preparation of a kit, a chip or a test strip for multiple detection of salmon and trout viruses.
It can be understood that the reagent of the present application is developed for multiplex detection of spring viraemia of carp virus, infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, infectious salmon anemia virus, salmon alphavirus and infectious pancreatic necrosis virus, and six salmon and trout viruses, and therefore, the reagent of the present application can be made into a corresponding multiplex detection kit or chip or test strip for a user. For example, based on the requirement of real-time fluorescent PCR, the probes in the reagent are modified by fluorescent groups and fluorescent quenching groups, so as to prepare a real-time fluorescent PCR kit for multiple detection of salmon and trout viruses; or the probes in the reagent are fixed on a gene chip to prepare the gene chip for the multiple detection of the salmon and trout viruses, or the probes are fixed on fluorescent coding microspheres and detected by using a liquid phase chip in an implementation mode of the reagent, and the detection method can be specifically determined according to an actually adopted detection method and is not specifically limited herein.
In yet another aspect, the present application discloses a kit for multiplex detection of salmon and trout viruses, comprising the reagents of the present application.
Preferably, the kit of the present application further comprises reagents for performing at least one of reverse transcription, PCR amplification and liquid-phase chip detection.
It is understood that the kit serves to facilitate the multiplex detection of the salmon and trout viruses, and therefore, all relevant reagents can be considered to be put into the kit, but the kit can only contain components directly related to the reagents, such as fluorescent coded microspheres related to a liquid phase chip and the like, in combination with the cost and the convenience in storage and transportation.
The application further discloses a chip for multiple detection of salmon and trout viruses, which comprises a solid-phase carrier and a specific probe set fixed on the solid-phase carrier. The specific probe set is actually the first to sixth probes in the reagent of the present application, and will not be described in detail herein.
Preferably, the chip is a liquid phase chip, the solid phase carrier is a fluorescent coding microsphere, and the first probe to the sixth probe are respectively fixed on the fluorescent coding microspheres with different numbers.
It is understood that the liquid phase chip is a detection method specifically used in one implementation mode of the present application, and in view of the specificity of the primers and probes of the present application, it is not excluded that other detection methods may be used, for example, immobilization of probes on a glass carrier of a conventional gene chip, etc.
The application also discloses a fluorescence coding microsphere reagent for detecting the salmon and trout virus multiple liquid phase chip, wherein the fluorescence coding microsphere reagent consists of a first group of fluorescence coding microspheres, a second group of fluorescence coding microspheres, a third group of fluorescence coding microspheres, a fourth group of fluorescence coding microspheres, a fifth group of fluorescence coding microspheres and a sixth group of fluorescence coding microspheres; the groups of fluorescent coding microspheres have different numbers, a first probe is coupled on the first group of fluorescent coding microspheres, a second probe is coupled on the second group of fluorescent coding microspheres, a third probe is coupled on the third group of fluorescent coding microspheres, a fourth probe is coupled on the fourth group of fluorescent coding microspheres, a fifth probe is coupled on the fifth group of fluorescent coding microspheres, and a sixth probe is coupled on the sixth group of fluorescent coding microspheres. The first probe, the second probe, the third probe, the fourth probe, the fifth probe, and the sixth probe are also the first probe to the sixth probe in the reagent of the present application, and will not be described in detail herein.
The fluorescent coding microsphere reagent can be independently sold as a product as an independent component, and is matched with the six pairs of specific primers to realize the multi-liquid chip detection of the six salmon and trout viruses.
The beneficial effect of this application lies in:
the reagent for multiple detection of the salmon and trout viruses can carry out multiple detection on six common salmon and trout viruses with great harm, is simple and convenient to use, shortens the detection time of the six salmon and trout viruses, improves the detection efficiency and quality, prevents the spread of the disease condition by quickly detecting and controlling the six salmon and trout viruses, and has great significance on ensuring the production safety to the maximum extent.
Drawings
FIG. 1 is a graph showing the results of electrophoresis of amplification products obtained by RT-PCR using six salmon and trout viral RNAs as templates in the examples of the present application.
Detailed Description
With the rise and development of salmon and trout culture, the loss of salmon and trout epidemic diseases to the culture industry is more and more, so that multiple detection reagents are designed for six main epidemic disease viruses of the salmon and trout through a large amount of research, and a corresponding multiple detection method is established, so that the viruses causing the epidemic diseases can be rapidly and accurately obtained, and the prevention and control are convenient.
The reagent for multiple detection of salmon and trout viruses mainly comprises six pairs of specific primers, namely a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair and a sixth primer pair. The six pairs of specific primers are specific detection primers respectively designed for SVCV, IHNV, VHSV, ISAV, SAV and IPNV, and the six salmon and trout viruses. In an improved scheme, the application also designs a pair of universal primers for amplifying the amplification products of six pairs of specific primers again, so that the content of the detection target is further improved. Furthermore, six specific probes are designed aiming at target areas of six pairs of specific primers, and the six specific probes are matched with the six pairs of specific primers for use, so that the specificity and the sensitivity of detection are improved. In one implementation mode of the application, six specific probes are specifically combined with a liquid chip, the six specific probes are fixed on six groups of fluorescent coding microspheres with different numbers, and the liquid chip is used for carrying out multiple detection on six salmon and trout viruses.
In design, the method for simultaneously detecting six salmon and trout viruses is established by using Locked Nucleic Acid (LNA) labels to adjust the dissolution temperature of a specific probe, performing Target fragment amplification through Target enriched multiplex polymerase chain reaction (Tem-PCR), and combining a Biotin-Avidin System (Biotin-Avidin-System, BAS) signal reaction amplification System and an xMAP liquid chip platform. In this application, the six salmon and trout viruses refer to SVCV, IHNV, VHSV, ISAV, SAV and IPNV.
Compared with the prior art, the reagent and the liquid chip detection method for six salmon and trout viruses based on the reagent have the following advantages:
(1) high flux and less sample requirement
In synchronization withThe detection target quantity and the detection sample quantity both represent high-flux detection capability, and by adopting the reagent and the liquid-phase chip detection method, the target genes of various viruses can be synchronously detected and identified, and a large number of samples can be continuously, automatically and rapidly detected. The number of target molecules which can be combined by a single microsphere reaches 106Therefore, only a few microliters of nucleic acid samples are required to detect six salmon and trout viruses.
(2) Quick reaction and high sensitivity
The liquid-phase chip detection method has the advantages that the reaction environment is liquid phase, the probes fixed on the fluorescent coding microspheres and a sample to be detected react in solution, and the collision probability and speed between the probes and the sample to be detected are increased by more than 10 times compared with a solid-phase chip reaction mode, so that the reaction speed and sensitivity can be improved. In an implementation mode of the application, a laser detection technology and a digital information processing technology are introduced, so that the characteristics of rapidness and sensitivity in detection are more prominent.
(3) Detection specificity enhancement by locked nucleic acid labeled probes
The locked nucleic acid marker increases the complementary pairing capacity of the primer or the probe to the template, can improve the Tm value of the primer or the probe with a shorter conserved region, ensures the same renaturation temperature and hybridization efficiency between the primer or the probe, and effectively avoids the cross hybridization between microspheres marked by different detection objects.
(4) Simple and flexible operation
The reagent and the detection method can simultaneously detect six salmon and trout viruses such as SVCV, IHNV, VHSV, ISAV, SAV, IPNV and the like by only one test, and can also freely increase and decrease the six salmon and trout viruses according to actual conditions without mutual interference; for example, when only one, two, three, four or five viruses are definitely required to be detected, only corresponding specific primers and corresponding specific probes or specific probes are required to be coupled with fluorescent coding microspheres, six pairs of specific primers or six specific probes are required to be coupled with fluorescent coding microspheres, the specific primers or six specific probes are not completely used, and the specific primers, six specific probes or six specific probes are freely combined according to specific detection requirements to realize the detection of the double, triple, quadruple or quintuple trout viruses.
(5) Simplified process
According to the liquid-phase chip detection method, after PCR amplification, products do not need to be processed, and can be directly hybridized with the fluorescent coding microspheres coupled with the specific probes, so that steps and time are saved, and the process is simplified.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
1. Materials and methods
1.1 Main test materials
The main test materials used in this example include Qiagen One Step RT-PCR Kit and EZ1 Virus miniKit2.0, both available from Qiagen, phycoerythrin (SAPE) from Invitrogen, USA. Liquichip carboxybead is available from Luminex corporation, Luminex catalysis bead mix and Luminex control bead kit is available from Qiagen corporation.
1.2 Main instruments
A small, fully automated nucleic acid extraction Workstation (BioRobot EZ1 Workstation) was purchased from Qiagen, a high speed desktop refrigerated centrifuge (Allegra 64R centrifuge) was purchased from Beckman, and a liquid phase chip detection Workstation (liquid 200) was purchased from Qiagen.
1.3 test Virus materials
The test materials of this example include an Infectious Hematopoietic Necrosis Virus (IHNV) nucleic acid sample, an Infectious Pancreatic Necrosis Virus (IPNV) nucleic acid sample, an Infectious Salmon Anemia Virus (ISAV) nucleic acid sample, a Salmon Alphavirus (SAV) nucleic acid sample, a Viral Hemorrhagic Septicemia Virus (VHSV) nucleic acid sample, a carp Spring viraemia virus (Spring vira of SVcarp virus, SAV) nucleic acid sample, and a brocade Herpesvirus disease (Koi herpesvira virus, KHV) nucleic acid sample, a carp virus type II (Cyprinus herpesviridis II, Cytositi 2) nucleic acid sample, an Infectious bovine hemorrhagic fever virus (SGNV) nucleic acid sample, an Infectious bovine hemorrhagic fever virus (HCV) nucleic acid sample, an Infectious bovine hemorrhagic septicemia virus (HIV) nucleic acid sample, a porcine reproductive Herpesvirus virus II nucleic acid sample, an HIV sample, a porcine reproductive Herpesvirus virus IV nucleic acid sample, a porcine reproductive necrosis virus (SGNV) nucleic acid sample, a porcine reproductive hemorrhagic septicemia virus (HCV) nucleic acid sample, a porcine reproductive Herpesvirus virus (HIV) nucleic acid sample, a porcine reproductive Herpesvirus sample, a porcine reproductive virus (HCV virus II, a porcine reproductive necrosis virus IV nucleic acid sample, a porcine reproductive necrosis virus (HCV) nucleic acid sample, a porcine reproductive necrosis virus IV sample, a porcine reproductive necrosis virus sample, a porcine, Canine Coronavirus (CCV) nucleic acid samples and rotavirus (HRV for short) nucleic acid samples. The nucleic acid samples are provided and stored by Shenzhen customs animal and plant inspection and quarantine technical center.
1.4 design of primers and probes
In the embodiment, according to the whole genome sequences of six salmon and trout viruses including SVCV, IHNV, VHSV, ISAV, SAV and IPNV, through comparison and analysis of software such as DNAMAN and DNA STAR, six pairs of specific primer pairs are designed for six salmon and trout viruses by screening conserved genes, and six specific probes are designed according to amplification target regions of the six pairs of specific primer pairs. In addition, in order to increase the amount of the detection target, in this example, a universal sequence independent of the detection target is further designed upstream and downstream of the six pairs of specific primer pairs, and a pair of universal primers is designed based on the universal sequence.
The six pairs of specific primers of this example include a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair and a sixth primer pair, and the first to sixth primer pairs specifically amplify the spring viraemia of carp virus, the infectious hematopoietic necrosis disease virus, the viral hemorrhagic septicemia virus, the infectious salmon anemia virus, the salmon alphavirus and the infectious pancreatic necrosis disease virus in this order. The upstream and downstream primers of the first primer pair are respectively a sequence shown in Seq ID No.1 and a sequence shown in Seq ID No. 2; the upstream and downstream primers of the second primer pair are respectively a sequence shown in Seq ID No.3 and a sequence shown in Seq ID No. 4; the upstream primer and the downstream primer of the third primer pair are respectively a sequence shown in Seq ID No.5 and a sequence shown in Seq ID No. 6; the upstream primer and the downstream primer of the fourth primer pair are respectively a sequence shown in Seq ID No.7 and a sequence shown in Seq ID No. 8; the upstream primer and the downstream primer of the fifth primer pair are respectively a sequence shown in Seq ID No.9 and a sequence shown in Seq ID No. 10; the upstream and downstream primers of the sixth primer set are the sequence shown in Seq ID No.11 and the sequence shown in Seq ID No.12, respectively. The upstream and downstream primers of the universal primer set in this example were Seq ID No.13 and Seq ID No.14, respectively.
The six specific probes of the example comprise a first probe, a second probe, a third probe, a fourth probe, a fifth probe and a sixth probe; the first to sixth probes are probes specifically hybridizing to spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order; the first probe is the sequence shown in Seq ID No.15, the second probe is the sequence shown in Seq ID No.16, the third probe is the sequence shown in Seq ID No.17, the fourth probe is the sequence shown in Seq ID No.18, the fifth probe is the sequence shown in Seq ID No.19, and the sixth probe is the sequence shown in Seq ID No. 20.
The six specific probes designed by the embodiment couple the fluorescent coding microspheres for the liquid phase chip, so that the 5' end of each probe is added with a C12 arm and is subjected to amination modification, the probe has enough extension arms, the probe can be coupled with the fluorescent coding microspheres better, and the sequence integrity of the probe is ensured. The six specific probes in the embodiment must be conserved to ensure that the missed detection cannot occur among different genes of the same virus; therefore, on the basis of ensuring the specificity of six specific probes, this example regulates the dissolution temperature of the probes by locking the nucleic acid label so that the Tm of the probes is maintained at 70 ℃. + -. 1 ℃. The primers and probes finally designed in this example were all synthesized by Biotechnology engineering (Shanghai) GmbH, as shown in Table 1.
TABLE 1 specific primer pairs and probes
In Table 1, "Bio" represents a biotin label, "NH 2" amino modified, "C12" represents a C12 extender arm, and the "+" symbol represents that the base at the 3' end is a base modified with a locked acid.
1.5 coupling of nucleic acid probes to fluorescent-encoded microspheres
The designed six specific probes are respectively coupled with the fluorescent coding microspheres (Luminex) with different numbers, and the numbers of the six specific probes and the corresponding fluorescent coding microspheres are shown in Table 2.
TABLE 2 numbering of specific probes and corresponding fluorescent-encoded microspheres
| |
1 | 2 | 3 | 4 | 5 | 6 |
| Microsphere numbering | 33 | 34 | 36 | 37 | 42 | 43 |
| Probe needle | SVCVProbe | ISAVProbe | VHSVProbe | IHNVProbe | SAVProbe | IPNVProbe |
In table 2, "microsphere number" refers to the number of the fluorescently encoded microsphere, e.g., "33" refers to the 33-numbered fluorescently encoded microsphere; "Probe" refers to a Probe to which a fluorescently encoded microsphere is coupled, e.g., "SVCV Probe" refers to a Probe specific for SVCV.
The method for coupling the specific probe and the fluorescent coding microspheres comprises the steps of balancing all reagents to room temperature for at least 30 min; resuspending and uniformly mixing the fluorescent coding microsphere stock solution by using ultrasonic waves or an oscillation method, immediately taking 20 mu L of the fluorescent coding microsphere stock solution from a storage tube into a 1.5mL polypropylene microcentrifuge tube, and centrifuging for 5min at 10000 r/min; discarding the supernatant, adding 50 μ L of 0.1M MES (pH4.5), shaking, mixing, adding 0.2nmol of specific probe, and mixing; adding 10 μ L of freshly prepared 10mg/mL EDC solution, uniformly mixing, incubating for 30min at room temperature in a dark place, and repeatedly adding the same amount of EDC again to continue incubation for 30 min; adding 1.0mL of Tween-20 (0.02% w/v), mixing uniformly, washing, centrifuging at 10000r/min for 10min, and precipitating the fluorescent coding microspheres; discarding the supernatant, adding 1.0mL of SDS (0.1% w/v), mixing uniformly, washing, centrifuging at 10000r/min for 10min again, and precipitating the fluorescent coding microspheres; adding 0.1M MES (pH4.5), counting the prepared fluorescent coding microspheres by adopting a blood cell counting method, calculating the concentration of the fluorescent coding microspheres, diluting the fluorescent coding microspheres into 1250 fluorescent coding microspheres/mu L by using the MES, and storing the microspheres at the temperature of 2-8 ℃ in a dark place for later use.
1.6 target sequence enrichment RT-PCR reaction
Respectively taking nucleic acid samples of SVCV, IHNV, VHSV, ISAV, SAV and IPNV salmon and trout viruses as templates, and carrying out amplification according to the following method, wherein the reaction system is as follows: 5 × RT-PCR buffer 10 μ L, dNTP mix 2 μ L, upstream and downstream universal primer mixture 1.5 μ L, upstream and downstream specific primers 0.75 μ L each, RNase inhibitor 1 μ L, 5Q-solution 10 μ L, template 5 μ L, and make up 50 μ L with Depc water.
The reaction conditions are as follows: 30min at 50 ℃ and 15min at 95 ℃; then 15 cycles were entered: 30s at 94 ℃, 1min at 52 ℃ and 1min at 72 ℃; after the end of 15 cycles, 6 cycles were entered: 30s at 94 ℃ and 30s at 70 ℃ for 1 min; and after 6 cycles are finished, 35 cycles are entered: 94 ℃ for 20s, 55 ℃ for 30s, and 72 ℃ for 20 s; after the circulation, the extension was carried out at 72 ℃ for 10 min.
After RT-PCR amplification is finished, agarose gel electrophoresis is adopted to carry out preliminary judgment on an amplification product, and whether a target sequence meeting the expectation is amplified or not is detected.
1.7 hybridization
A50. mu.L volume of hybridization reaction mixture included 33. mu.L of 1.5 XTMAC nucleic acid detection buffer, 2. mu.L each of six specific probe-coupled microspheres, and 5. mu.L of the product of the "1.6 target sequence-enriched RT-PCR reaction". In addition, 5. mu.L of TE solution (pH8.0) was used as a blank in place of 5. mu.L of the target sequence-enriched product.
Gently and repeatedly pumping, uniformly mixing, covering the reaction chamber with a cover, and carrying out the following reactions in a PCR instrument: 5min at 95 ℃ and 30min at 50 ℃; then, 10000r/min was centrifuged for 3min, and during centrifugation, SA-PE was diluted to 2. mu.g/mL with 1 XTMAC hybridization solution to prepare a report mixture. After centrifugation, the supernatant was discarded and 75. mu.L of the reporter mixture was added, gently whipped and mixed, and incubated on a PCR instrument at 52 ℃ for 5 min. According to the instrument detection steps recommended by Luminex company, an instrument is arranged to detect only the correspondingly coded fluorescent coding microspheres at each time, a sample loading heating ceramic base plate is preheated to 52 ℃, and then a sample is loaded on the machine for detection.
1.8 liquid chip detection
And (3) resuspending and uniformly mixing the fluorescence coding microspheres coupled with the probes by using an oscillation or ultrasonic method, respectively taking 10 mu L of the prepared fluorescence coding microspheres, and further diluting each coded fluorescence coding microsphere into 200 fluorescence coding microspheres/mu L by using 1.5 times TMAC hybridization solution. Adding 33 mu L of mixed fluorescent coding microsphere working solution into each sample hole and each background hole; for each background well, 17 μ LTE (ph8.0) was added as a blank control; for each sample well, 5. mu.L and 12. mu.L TE (pH8.0) of the purified PCR product were added, and after gently and repeatedly pipetting and mixing, the reaction was covered with a cap, and the following reaction was performed in a PCR apparatus: centrifuging at 95 deg.C for 5min, 52 deg.C for 15min and 10000r/min for 3min, and diluting SA-PE to 2 μ g/mL with 1 × TMAC hybridization solution during centrifugation to obtain report mixed solution. After centrifugation, the supernatant was discarded and 75. mu.L of the reporter mixture was added, gently whipped several times and mixed, and incubated on a PCR instrument at 52 ℃ for 5 min. According to the instrument detection steps recommended by Luminex company, an instrument is arranged to detect only the correspondingly coded fluorescent coding microspheres at each time, a sample loading heating ceramic base plate is preheated to 52 ℃, and then a sample is loaded on the machine for detection.
According to the judgment standard recommended by Luminex company, when the number of each fluorescent coding microsphere is not less than 20 and the background blank fluorescence intensity is not higher than 300, the experiment is proved to be true, and the result is judged.
The liquid phase chip qualitative ratio result (LQRR) is equal to the ratio of the corrected Median Fluorescence Intensity (MFI) of the sample to the mean MFIB of the blank MFI, i.e., LQRR is MFIs/MFIB. If LQRR is more than or equal to 3, determining as a positive sample; if the LQRRs are more than or equal to 2 and less than or equal to 3, determining the system to be suspicious; if LQRR <2, then the result is judged to be negative.
1.9 detection of specificity of liquid chip detection System
According to the aforementioned method, this example was performed by performing "1.6 target sequence enrichment RT-PCR reaction" using IHNV nucleic acid sample, IPNV nucleic acid sample, ISAV nucleic acid sample, SAV nucleic acid sample, VHSV nucleic acid sample, SVCV nucleic acid sample, KHV nucleic acid sample, CyHV-2 nucleic acid sample, SGIV nucleic acid sample, RSIV nucleic acid sample, EHNV nucleic acid sample, CCV nucleic acid sample and HRV nucleic acid sample as templates, and then performing liquid phase chip detection according to "1.7 hybridization" and "1.8 liquid phase chip detection" for determining the specificity of the primer, probe and liquid phase chip detection methods in this example.
1.10 liquid-phase chip detection system sensitivity verification
IHNV nucleic acid samples, IPNV nucleic acid samples, ISAV nucleic acid samples, SAV nucleic acid samples, VHSV nucleic acid samples and SVCV nucleic acid samples are respectively diluted by 10 times in a gradient way, in the example, each nucleic acid sample is respectively diluted 5 times, then the nucleic acid samples diluted in the gradient way are subjected to 1.6 target sequence enrichment RT-PCR reaction, and liquid phase chip detection is carried out according to 1.7 hybridization and 1.8 liquid phase chip detection, so as to test the sensitivity of the liquid phase chip detection method in the example.
2. Results of the experiment
2.1RT-PCR amplification product detection
The RT-PCR amplification products were detected by agarose gel electrophoresis, and the results are shown in FIG. 1. In FIG. 1, the molecular weight standard is Marker DL 3000, SVCV in lane 1, IHNV in lane 2, VHSV in lane 3, ISAV in lane 4, SAV in lane 5, IPNV in lane 6, and blank control in lane 7. The results in FIG. 1 show that the RT-PCR amplification has the expected target sequences for IHNV, IPNV, ISAV, SAV, VHSV, and SVCV, but no target sequence for the blank control, indicating that the RT-PCR amplification of this example can effectively amplify the target sequence.
2.2 specific detection of liquid chip detection System
In this example, IHNV, IPNV, ISAV, SAV, VHSV, SVCV, KHV, CyHV-2, SGIV, RSIV, EHNV, CCV, and HRV nucleic acid samples were subjected to RT-PCR amplification, and liquid phase chip detection was carried out using the respective amplification products as templates, and the results are shown in Table 3.
TABLE 3 detection results of specificity of liquid phase chip
In Table 3, "SVCV-33" refers to the results of detection of fluorescent-encoded microsphere # 33 coupled to SVCV-specific probes.
The detection results in Table 3 show that the detection method of the liquid phase chip of this example has detection signals only for IHNV nucleic acid sample, IPNV nucleic acid sample, ISAV nucleic acid sample, SAV nucleic acid sample, VHSV nucleic acid sample, SVCV nucleic acid sample, and no detection signals for other negative samples tested; furthermore, each primer pair and probe will only detect a signal from its corresponding target nucleic acid sample, but will not have a signal from other non-targets, e.g., "SVCV-33" will only detect a stronger signal from the SVCV sample, while several others, e.g., IHNV, IPNV, ISAV, SAV, VHSV, will not have a signal. The primer and the probe of the embodiment have good specificity, can accurately and effectively detect the six salmon and trout viruses of SVCV, IHNV, VHSV, ISAV, SAV and IPNV, and can realize the multiple detection of the six salmon and trout viruses at one time.
2.3 sensitivity verification of liquid chip detection System
IHNV, IPNV, ISAV, SAV, VHSV, SVCV viral nucleic acids were serially diluted 10-fold each, and the results of the sensitivity detection were shown in Table 4 for a total of 5 dilutions.
TABLE 4 sensitivity test results
In Table 4, "SVCV-33" refers to the detection result of fluorescent-encoded microsphere No. 33 coupled with SVCV-specific probe, "LQRR 100"refers to the result of detection of nucleic acid sample without dilution," LQRR10-1"means the result of the test performed with 10-fold gradient dilution," LQRR10-2"means the result of the test performed 100-fold gradient dilution," LQRR10-3"means the result of the test performed with 1000-fold gradient dilution," LQRR10-4"means the result of the test by 10000-fold gradient dilution," LQRR10-5"means the result of measurement by gradient dilution 100000 times.
The results in Table 4 show that the lower limit of detection of ISAV nucleic acid samples, IPNV, IHNV, VHSV and SAV nucleic acid samples, was 100-fold gradient dilution, the lower limit of detection was 1000-fold gradient dilution and the lower limit of detection was 10000-fold gradient dilution. According to the conversion of the test result of the nucleic acid concentration, the lowest detection limit of the SVCV is 0.14 pg/mu L; the lowest detection limit of IHNV is 0.1pg/μ L; the lowest detection limit of VHSV is 1.6pg/μ L; the lowest detection limit of ISAV is 2 pg/mu L; the lowest detection limit of SAV is 0.9pg/μ L; the minimum detection limit for IPNV is 0.4 pg/. mu.L.
2.3 clinical applications
The primers, probes and liquid chip detection method of the example are adopted to test the blind sample provided by the Danish fish disease reference laboratory. The detection results show that 1 SVCV, 1 IHNV, 1 VHSV, 1 ISAV and 1 SAV are detected, and the detection results are consistent with the actual conditions.
In addition, 35 rainbow trout samples collected in China are tested, and the results show that 5 IHNV samples and 1 IHNV and IPNV co-infection sample are detected, and the results are consistent with the actual conditions.
The results show that the primer, the probe and the liquid chip detection method can effectively detect actual samples and can realize multiple detection of six salmon and trout viruses including SVCV, IHNV, VHSV, ISAV, SAV and IPNV at one time.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Shenzhen customs animal and plant inspection and quarantine technical center
<120> reagent for multiple detection of salmon and trout viruses and application thereof
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Claims (7)
1. A reagent for multiple detection of salmon and trout viruses, which is characterized by comprising the following components in percentage by weight: the kit comprises a specific primer group, a specific probe group and a universal primer pair, wherein the specific primer group comprises a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair and a sixth primer pair;
the first to sixth primer pairs are primer pairs for specifically amplifying spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order;
the upstream primer and the downstream primer of the first primer pair are respectively a sequence shown in Seq ID No.1 and a sequence shown in Seq ID No. 2; the upstream primer and the downstream primer of the second primer pair are respectively a sequence shown in Seq ID No.3 and a sequence shown in Seq ID No. 4; the upstream primer and the downstream primer of the third primer pair are respectively a sequence shown by Seq ID No.5 and a sequence shown by Seq ID No. 6; the upstream primer and the downstream primer of the fourth primer pair are respectively a sequence shown in Seq ID No.7 and a sequence shown in Seq ID No. 8; the upstream primer and the downstream primer of the fifth primer pair are respectively a sequence shown in Seq ID No.9 and a sequence shown in Seq ID No. 10; the upstream primer and the downstream primer of the sixth primer pair are respectively a sequence shown in Seq ID No.11 and a sequence shown in Seq ID No. 12;
Seq ID No.1：5’-CAGGCCACGTATTGTCATGCTGCCAAATCACCATACTCAACAATAA-3’
Seq ID No.2：5’-TTCTTGGCGTTATGTCGCTGGAGTCAATCTCATCAGGCTGTCTTG-3’
Seq ID No.3：5’-CAGGCCACGTATTGTCATGCAGAGCCAAGGCACTGTGCG-3’
Seq ID No.4：5’-TTCTTGGCGTTATGTCGCTGTTCTTTGCGGCTTGGTTGA-3’
Seq ID No.5：5’-CAGGCCACGTATTGTCATGCAAACTCGCAGGATGTGTGCGTCC-3’
Seq ID No.6：5’-TTCTTGGCGTTATGTCGCTGTCTGCGATCTCAGTCAGGATGAA-3’
Seq ID No.7：5’-CAGGCCACGTATTGTCATGCCTACACAGCAGGATGCAGATGT-3’
Seq ID No.8：5’-TTCTTGGCGTTATGTCGCTGCAGGAGCCGGAAGTCGAT-3’
Seq ID No.9：5’-CAGGCCACGTATTGTCATGCCCGGCCCTGAACCAGTT-3’
Seq ID No.10：5’-TTCTTGGCGTTATGTCGCTGGTAGCCAAGTGGGAGAAAGCT-3’
Seq ID No.11：5’-CAGGCCACGTATTGTCATGCGAGGTCTCAGACTCCGGAAGTG-3’
Seq ID No.12：5’-TTCTTGGCGTTATGTCGCTGTCCAGCGCCGTCTGGTT-3’；
the specific probe group comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe and a sixth probe;
the first to sixth probes are probes specifically hybridizing to spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order;
the first probe is a sequence shown in Seq ID No.15, the second probe is a sequence shown in Seq ID No.16, the third probe is a sequence shown in Seq ID No.17, the fourth probe is a sequence shown in Seq ID No.18, the fifth probe is a sequence shown in Seq ID No.19, and the sixth probe is a sequence shown in Seq ID No. 20;
Seq ID No.15：5’-TTGTTGCTGCATTGTGCCGCTCC-3’
Seq ID No.16：5’-TGAGACTGAGCGGGACA-3’
Seq ID No.17：5’-TAGAGGGCCTTGGTGATCTTCTG-3’
Seq ID No.18：5’-CATCGTCGCTGCAGTTC-3’
Seq ID No.19：5’-CTGGCCACCACTTCGA-3’
Seq ID No.20：5’-CTTCTAGTCTGCTTCCCGGGTGCACC-3’；
in the second probe having the sequence shown in Seq ID No.16, the third base, the seventh base and the eighth base are all acid-locked modified bases;
in the third probe with the sequence shown in Seq ID No.17, the seventh base and the eighth base are both acid-locked modified bases;
in the fourth probe having the sequence represented by Seq ID No.18, the fifth base, the sixth base, the seventh base and the twelfth base are all acid-locked modified bases;
in the fifth probe having the sequence represented by Seq ID No.19, the fifth base, the sixth base and the eighth base are all acid-locked modified bases;
in the first to sixth probes, the 5' end of each probe has a carbon arm and an amination modification; the carbon arm is a C6 arm or a C12 arm;
the upstream primer and the downstream primer of the universal primer pair are respectively a sequence shown in Seq ID No.13 and a sequence shown in Seq ID No. 14;
Seq ID No.13：5’-CAGGCCACGTATTGTCATGC-3’
Seq ID No.14：5’-TTCTTGGCGTTATGTCGCTG-3’
in the universal primer pair, the 5' segment of the sequence primer shown in Seq ID No.14 has a biotin label, and the fifth base and the thirteenth base in the sequence are both internally biotin-labeled deoxythymine;
the salmon and trout viruses include spring viraemia of carp virus, infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, infectious salmon anemia virus, salmon alphavirus, and infectious pancreatic necrosis virus.
2. Use of the reagent according to claim 1 in the preparation of a kit, chip or strip for the multiplex detection of salmon and trout viruses.
3. A kit for multiple detection of salmon and trout viruses, which is characterized in that: comprising the reagent of claim 1.
4. The kit of claim 3, wherein: and reagents for performing at least one of reverse transcription, PCR amplification and liquid chip detection.
5. A chip for multiple detection of salmon and trout viruses, which is characterized in that: the kit comprises a solid phase carrier and a specific probe set fixed on the solid phase carrier, wherein the specific probe set comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe and a sixth probe;
the first to sixth probes are probes specifically hybridizing to spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order;
the first probe is a sequence shown in Seq ID No.15, the second probe is a sequence shown in Seq ID No.16, the third probe is a sequence shown in Seq ID No.17, the fourth probe is a sequence shown in Seq ID No.18, the fifth probe is a sequence shown in Seq ID No.19, and the sixth probe is a sequence shown in Seq ID No. 20;
in the second probe having the sequence shown in Seq ID No.16, the third base, the seventh base and the eighth base are all acid-locked modified bases;
in the third probe with the sequence shown in Seq ID No.17, the seventh base and the eighth base are both acid-locked modified bases;
in the fourth probe having the sequence represented by Seq ID No.18, the fifth base, the sixth base, the seventh base and the twelfth base are all acid-locked modified bases;
in the fifth probe having the sequence represented by Seq ID No.19, the fifth base, the sixth base and the eighth base are all acid-locked modified bases;
in the first to sixth probes, the 5' end of each probe has a carbon arm and an amination modification; the carbon arm is a C6 arm or a C12 arm.
6. The chip of claim 5, wherein: the chip is a liquid phase chip, the solid phase carrier is a fluorescent coding microsphere, and the first probe to the sixth probe are respectively fixed on the fluorescent coding microspheres with different numbers.
7. A fluorescence coding microsphere reagent for detecting salmon and trout virus multiple liquid phase chips is characterized in that: the fluorescent coding microsphere reagent consists of a first group of fluorescent coding microspheres, a second group of fluorescent coding microspheres, a third group of fluorescent coding microspheres, a fourth group of fluorescent coding microspheres, a fifth group of fluorescent coding microspheres and a sixth group of fluorescent coding microspheres;
each group of fluorescent coding microspheres has different numbers, a first probe is coupled on the first group of fluorescent coding microspheres, a second probe is coupled on the second group of fluorescent coding microspheres, a third probe is coupled on the third group of fluorescent coding microspheres, a fourth probe is coupled on the fourth group of fluorescent coding microspheres, a fifth probe is coupled on the fifth group of fluorescent coding microspheres, and a sixth probe is coupled on the sixth group of fluorescent coding microspheres;
the first to sixth probes are probes specifically hybridizing to spring viraemia of carp virus nucleic acid, infectious hematopoietic necrosis virus nucleic acid, viral hemorrhagic septicemia virus nucleic acid, infectious salmon anemia virus nucleic acid, salmon alphavirus nucleic acid, and infectious pancreatic necrosis virus nucleic acid, in this order;
the first probe is a sequence shown in Seq ID No.15, the second probe is a sequence shown in Seq ID No.16, the third probe is a sequence shown in Seq ID No.17, the fourth probe is a sequence shown in Seq ID No.18, the fifth probe is a sequence shown in Seq ID No.19, and the sixth probe is a sequence shown in Seq ID No. 20;
in the second probe having the sequence shown in Seq ID No.16, the third base, the seventh base and the eighth base are all acid-locked modified bases;
in the third probe with the sequence shown in Seq ID No.17, the seventh base and the eighth base are both acid-locked modified bases;
in the fourth probe having the sequence represented by Seq ID No.18, the fifth base, the sixth base, the seventh base and the twelfth base are all acid-locked modified bases;
in the fifth probe having the sequence represented by Seq ID No.19, the fifth base, the sixth base and the eighth base are all acid-locked modified bases;
in the first to sixth probes, the 5' end of each probe has a carbon arm and an amination modification; the carbon arm is a C6 arm or a C12 arm.
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