Signal amplification method based on biotin and streptavidin

Abstract

The invention discloses a signal amplification method based on biotin and streptavidin, which amplifies a signal to be detected through the connection of biotin and streptavidin molecules so as to improve the sensitivity of nucleic acid detection. The multi-biotin and streptavidin molecule comprises a single-stranded nucleic acid molecule for identifying a linker molecule through hybridization and a carrier molecule for biotin labeling. Therefore, one target molecule can be connected with the connecting molecule, the carrier molecule, a plurality of biotins and corresponding markers so as to amplify a detected signal. A plurality of linker molecules are connected to different regions of the target molecule, so that a greater amplification effect can be obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of vital characteristic substances, and particularly to a signal amplification method based on biotin and streptavidin. Background Art

[0002] Vital characteristic substances include two major categories: DNA and RNA.

[0003] Taking DNA substances as an example, hepatitis B is a liver infection caused by the hepatitis B virus, which poses a great potential threat to the body and is a major health problem in the world and the most serious type of viral hepatitis. Although the implementation of the planned vaccination in the endemic areas has successfully reduced the prevalence of hepatitis B surface antigen (HBsAg, the serological marker of chronic infection), there are still 350 million virus carriers, 25% of whom will develop liver diseases, liver cirrhosis and primary hepatocellular carcinoma (HCC), and it is estimated that 600,000 people die from acute and chronic diseases caused by hepatitis B every year.

[0004] Markers in the blood can be used for the diagnosis of hepatitis B infection and the differential diagnosis of acute and chronic infections. These markers include the antigens and antibodies of the hepatitis B virus. There are three commonly used antigens of the hepatitis B virus - surface antigen (HBsAg), the current diagnosis of HBV infection is based on the immunological detection of surface antigen, core antigen (HBcAg) and e antigen (HBeAg) and their corresponding antibodies. Although these detections can be used for the primary screening of carriers, they cannot be used for the detection of infectious viruses in serum.

[0005] HBV-DNA is the most direct marker of virus replication. HBV is a small DNA virus, and its nuclear capsid contains a 3.2Kb partially double-stranded genome, and its replication is mediated by RNA. Measuring HBV-DNA in serum is an important tool for differentiating whether the virus replicates. Detecting the HBV-DNA marker in a blood specimen indicates active virus replication. In acute hepatitis, HBV-DNA appears soon after infection and disappears after the infection is cleared. In chronic hepatitis B, the level of HBV-DNA often continues to increase for many years and decreases when the immune system controls the virus. For chronic hepatitis B, PCR detection is particularly useful for some patients who are positive for HBsAg, anti-HBe and have active HBV replication.

[0006] 5-10% of the patients undergoing liver transplantation develop HBV-related chronic and fulminant liver diseases. HBV reinfection may be due to the direct reinfection of HBV virus particles derived from circulating blood, extrahepatic sites or both. Without preventive detection, the risk of HBV reinfection is almost 80%. Recurrent infection can lead to graft failure, retransplantation or death. Therefore, follow-up of HBV infection after liver transplantation is crucial for long-term survival. This requires more sensitive HBV-DNA detection.

[0007] Antiviral therapy is the only option for controlling and preventing the progression of chronic hepatitis B. Nucleoside analogs have been shown to be effective in controlling the disease process, but the occurrence of drug resistance limits the long-term efficacy. Lamivudine was the first nucleoside analog approved for the treatment of this disease. The annual incidence of lamivudine resistance is 14-24%, and the incidence over 4-5 years is 70%. Due to the occurrence of drug-resistant strains, it is becoming increasingly important to monitor patients comprehensively. Multiple mutations mainly occur at the active site, YMDD, in the HBV polymerase reverse transcriptase region. There are two main mutations associated with lamivudine resistance: rtM204V and rtM204I. Other mutations include rtL80V, rtI169T, rtV173L, rtL180M, rt181T, rtT184S, rtM240V / I / S, and rtQ215S.

[0008] Currently, HBV drug resistance detection is based on PCR methods, but there are two potential problems. First, false positives may occur due to DNA contamination. Second, the simultaneous detection of multiple mutations by PCR is very limited.

[0009] PCR is a method for directly and highly sensitively detecting the viral genome, but the risk of false positives due to contamination and the difficulty in developing quantitative detection methods may prevent its application. In addition, false negatives may be caused by suboptimal reaction conditions and incomplete denaturation of DNA, and errors in specimen collection and / or processing may also cause problems.

[0010] Taking RNA substances as an example, hepatitis C virus (HCV) is another major cause of liver-related morbidity and mortality. There are 170 million chronic infections caused by HCV globally, accounting for 3% of the world's population. The long-term risk of severe clinical disease is cirrhosis and liver cancer. Similar to HBV, HCV infection can cause acute and chronic hepatitis C, cirrhosis, and primary hepatocellular carcinoma (HCC). Studies have shown that compared with HBV, HCV has a closer relationship with HCC. 80% of HCC is caused by HCV, while 15% is caused by HBV. What is significantly different between HCV infection and HBV infection is that HCV infection is associated with fatty liver, liver-mediated diabetes, and B-cell lymphoma.

[0011] HCV is a single-stranded RNA virus with rapid spontaneous mutations. The mutation frequency is estimated to be 10^-2 per nucleotide per year, resulting in significant differences in the sequences of many isolated virus strains worldwide. This makes vaccine development difficult. Therefore, effective diagnosis of HCV is crucial for preventing the spread of hepatitis C, which will save medical resources and reduce medical expenses for HCV in the long run. Based on certain genetic differences and similarities, HCV can be divided into six main genotypes: 1, 2, 3, 4, 5, and 6, which are distributed in different countries. Genotype information is very important for HCV treatment as it can predict the treatment effect. For example, peginterferon plus ribavirin treatment for genotype 2 or 3 requires 24 weeks, while for genotype 1 it is 48 weeks.

[0012] Many countries have introduced "guidelines" for HCV treatment, which will play a positive role in improving the current diagnosis of hepatitis C. Early diagnosis of hepatitis C is crucial for preventing further transmission among high-risk populations and for clinicians to make rapid treatment decisions, as rapid treatment has been proven to be very effective for acute hepatitis C.

[0013] The infection of hepatitis C virus can be determined by detecting HCV antibodies in the blood. Radioimmunoassay and ELISA for anti-HCV C-100 antibodies are the main diagnostic methods for hepatitis C, but they have many drawbacks. First, the anti-C100 antibody appears late and cannot be detected during the 8-week window period after acute infection. Half of the patients with post-transfusion hepatitis C have seroconversion of anti-C100 for the first time 4 to 6 months after blood transfusion. Therefore, it is not suitable for routine laboratory diagnosis of acute hepatitis. Second, it is not sensitive, and a small number of hepatitis C patients cannot be detected for C-100 antibodies. Third, it is not specific, and some patients with chronic liver diseases have false positives due to their own immune factors. Since the anti-HCV core antibody appears earlier, second-generation anti-HCV antibody detection reagents are based on the C-22-C protein in the C region of HCV and the C-33-3 and C-100-3 proteins encoded by the non-structural region NS3, which improves the detection efficiency by

[0014] 25 - 30%, advancing the initial detection period by 16 - 42 days.

[0015] Due to various factors, including antigen mutation, a large number of genotypes, a long window period, etc., single antibody detection or antigen detection is not a reliable method in terms of the safety of blood donation screening and is not efficient in the early diagnosis of HCV infection. Currently, HCV-RNA detection is a more direct and reliable method for diagnosing the virus itself and its replication. Since the amount of HCV in the blood of HCV-infected individuals is limited, it is difficult to detect HCV-RNA by direct hybridization without amplification. HCV is an RNA virus. Before PCR amplification, it is necessary to first convert RNA into cDNA, and primers designed from the conserved region of the 5' non-coding region are used for reverse transcription and amplification. The amplified product is observed by electrophoresis, and the method is very sensitive. HCV-RNA / PCR can be used to detect HCV-RNA in the liver and serum, and HCV can be detected in as little as 1-2 weeks (after infection). Since HCV-RNA in the liver and serum appears earlier than antibodies, HCV-RNA can be detected in the liver and serum in many HCV-infected individuals before anti-HCV seroconversion occurs. When HCV-RNA is positive, it indicates virus replication; when HCV-RNA is negative, it indicates virus clearance. Therefore, RT-PCR can be used for the early diagnosis of HCV, screening of blood donors, and prediction of the prognosis of hepatitis C. However, the main disadvantages of RT-PCR detection of HCV are the need to prepare RNA and reverse-transcribe cDNA, and in addition, certain techniques are required, the reproducibility is limited, specific instrument equipment is needed, and there is a risk of contamination. Summary of the Invention

[0016] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies of the prior art and propose a highly sensitive detection technique. The technology of the present invention realizes the sensitive detection of nucleic acids through signal amplification rather than PCR-based template amplification technology. Since there is no need for template amplification, the inherent contamination problem of PCR is avoided. More importantly, PCR is only used for DNA amplification. For RNA molecules such as HCV RNA virus and many mRNA markers, PCR requires converting RNA into cDNA before amplification. Since the technology of the present invention can directly detect RNA molecules without reverse transcription and pre-separation of RNA for transcription, the operation steps are greatly simplified. In addition, different from PCR, the technology of the present invention has a wider linear detection range and better precision, does not require special instrument equipment for detection, does not require expensive pre-labeled dyes for quantitative determination, and the detection is simple and efficient.

[0017] The technical solution adopted by the present invention to solve the above technical problems is to design and manufacture a signal amplification method based on biotin and streptavidin, and amplify the signal to be detected by connecting a multi-biotin and streptavidin molecule to improve the sensitivity of nucleic acid detection. The multi-biotin and streptavidin molecule includes a single-stranded DNA molecule that hybridizes and recognizes a linker molecule and a carrier molecule for biotin and streptavidin labeling.

[0018] The carrier molecule is DNA, RNA, oligonucleotide, polynucleotide or polymer.

[0019] The carrier molecule is single-stranded or double-stranded.

[0020] The biotin on the carrier molecule is biotin hybridized and labeled by an enzymatic reaction or a chemical reaction.

[0021] The biotin-streptavidin labeling enables at least two biotins to be covalently or non-covalently linked to the carrier molecule.

[0022] The linker molecule is DNA, RNA or oligonucleotide.

[0023] The linker molecule is part of one or more synthetic molecules hybridized to the target molecule.

[0024] The target molecule is captured on a microplate or other solid phase.

[0025] In one embodiment, the target molecule is HBV-DNA.

[0026] In another embodiment, the target molecule is HCV-RNA.

[0027] Compared with the prior art, the signal amplification method based on biotin and streptavidin of the present invention can greatly improve the detection sensitivity of the detected signal and eliminate various technical defects existing in PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is the basic principle of the signal amplification method based on biotin and streptavidin of the present invention.

[0030] Figure 2 It is the working principle of the signal amplification method based on biotin and streptavidin of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention. Additionally, it should be noted that for the convenience of description, only the parts related to the present invention rather than all the structures are shown in the drawings. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0032] Reference to "embodiment" in this document means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the present invention. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0033] A signal amplification method based on biotin and streptavidin will be further described in detail below in conjunction with the best embodiments shown in the respective drawings.

[0034] As Figure 1 shown, the basic principle of the signal amplification method based on biotin and streptavidin of the present invention is that the target molecule 1 can be captured on a microplate 6 or other solid phase, and the target molecule 1 can be connected to a linker molecule 2, a multi-biotin molecule 4, and a corresponding label 5 to achieve the detection of the target molecule 1.

[0035] As Figure 2 shown, the working principle of the signal amplification method based on biotin and streptavidin of the present invention is that the target molecule 1 can be captured on a microplate 6 or other solid phase, and the target molecule 1 can be connected to a linker molecule 2, a carrier molecule 3, the biotin molecules 4 carried on each carrier molecule 3, and a corresponding label 5 to achieve the amplified detection of the signal of the target molecule 1. It should be noted that the numbers in this figure are only used to illustrate the working principle of the present invention and do not constitute a limitation on the method of the present invention.

[0036] Among them, the linker molecule 2 can be DNA, RNA, or oligonucleotide, and it is a part of one or more synthetic molecules hybridized with the target molecule 1.

[0037] The carrier molecule 3 is DNA, RNA, oligonucleotide, polynucleotide, or polymer. It can be single-stranded or double-stranded, and it can be hybridized and labeled with biotin 4 through an enzymatic reaction or a chemical reaction. This biotin labeling enables at least two biotins 4 to be covalently or non-covalently connected to the carrier 3.

[0038] Thus, a target molecule 1 can be linked to multiple linker molecules 2, multiple carrier molecules 3, multiple biotin molecules 4, and corresponding markers 5, amplifying the detected signal. This technique is called the signal amplification method based on biotin and streptavidin (MBSA).

[0039] Taking the detection of HBV-DNA as an example, the signal amplification method based on biotin and streptavidin of the present invention amplifies the reporter signal rather than amplifying the target sequence. Therefore, it avoids the inescapable errors brought about during the extraction and amplification of the target sequence. In addition, for all known drug-resistant mutation sites of HBV, they can all be equally determined based on the MBSA target probe sequence. The technical and practical features of MBSA contribute to its routine clinical use. For serum and plasma specimens, no specimen preparation process is required, and for specimens with potential contamination, no special treatment process is needed. Applying the MBSA technique can accurately determine the viral load, omitting the cumbersome steps of nucleic acid extraction and reducing the possibility of cross-contamination.

[0040] The signal amplification method based on biotin and streptavidin of the present invention is similar to the sandwich ELISA method. In this method, after the DNA of the patient's serum is denatured, it can be directly added to the detection carrier, such as a 96-well microplate. The HBV-DNA is specifically captured by the oligonucleotides pre-coated in the microplate. After capture, the HBV-DNA molecule is incubated with the oligonucleotides in the test specimen, and then incubated with multiple biotin molecules. Thus, multiple rather than single streptavidin-horseradish peroxidase can bind to the captured HBV-DNA molecule, forming an amplification of the detection signal. Therefore, HBV-DNA can be highly sensitively detected.

[0041] Taking the detection of HCV-RNA as an example, the signal amplification method based on biotin and streptavidin of the present invention determines the HCV-RNA level by amplifying the reporter signal rather than amplifying the target sequence. Therefore, it avoids the inescapable errors brought about during the extraction and amplification of the target sequence. The target probe designed by the MBSA technique can equally quantify all known HCV genotypes. Therefore, this method can equally quantify all RNA transcripts including the sequences of 6 major HCV genotypes. The technical and practical features of MBSA contribute to its routine clinical use. For serum and plasma specimens, no specimen preparation process is required, and for specimens with potential contamination, no special treatment process is needed. Applying the MBSA technique can accurately determine the viral load, omitting the cumbersome steps of nucleic acid extraction and reducing the possibility of cross-contamination.

[0042] The signal amplification method based on biotin and streptavidin of the present invention is similar to the sandwich ELISA method. After the nucleic acid in the patient's serum is denatured, it can be directly added to a 96-well plate. HCV-RNA is specifically captured by the oligonucleotides pre-coated in the microplate. After capture, the HCV-RNA molecule is incubated with the oligonucleotides in the test specimen, and then incubated with multiple biotin molecules. Multiple rather than single streptavidin-horseradish peroxidase binds to the captured HCV-RNA molecule, so HCV-RNA can be detected with high sensitivity.

[0043] In summary, the signal amplification method based on biotin and streptavidin of the present invention generally includes three incubation steps:

[0044] 1. Incubate the specimen with the detection oligonucleotides;

[0045] 2. Incubate with multiple biotin molecules;

[0046] 3. Incubate with streptavidin-horseradish peroxidase.

[0047] It can be seen that the signal amplification method based on biotin and streptavidin of the present invention is as simple as the ELISA method, but can detect nucleic acid molecules with high sensitivity. Its advantages include, but are not limited to: First, there is no specimen pretreatment. The specimen is directly added to the plate wells, which not only simplifies the steps but also avoids experimental errors; Second, no special instruments are required, and every conventional diagnostic laboratory can perform it; Third, the detection cost is relatively low.

[0048] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A signal amplification method based on biotin and streptavidin, characterized in that Comprising: Amplifying a signal to be detected through the linkage of a multi-biotin molecule to improve the sensitivity of nucleic acid detection, where the multi-biotin molecule includes a single-stranded DNA molecule that hybridizes to recognize a linker molecule and a carrier molecule for biotin labeling.

2. The signal amplification method based on biotin and streptavidin according to claim 1, wherein The carrier molecule is DNA, RNA, oligonucleotide, polynucleotide or polymer.

3. The signal amplification method based on biotin and streptavidin according to claim 2, characterized in that, The carrier molecule is single-stranded or double-stranded.

4. The signal amplification method based on biotin and streptavidin according to claim 3, wherein The biotin on the carrier molecule is biotin labeled by hybridization through an enzymatic reaction or a chemical reaction.

5. The signal amplification method based on biotin and streptavidin according to claim 4, wherein The biotin labeling covalently or non-covalently links at least two biotins to the carrier.

6. The signal amplification method based on biotin and streptavidin according to claim 1, wherein, The linker molecule is DNA, RNA or oligonucleotide.

7. The signal amplification method based on biotin and streptavidin according to claim 6, wherein The linker molecule is part of one or more synthetic molecules that hybridize to the target.

8. The signal amplification method based on biotin and streptavidin according to claim 7, wherein, The target molecule is captured on a microplate or other solid phase.

9. The signal amplification method based on biotin and streptavidin according to claim 8, characterized in that, The target molecule is HBV-DNA.

10. The signal amplification method based on biotin and streptavidin according to claim 8, characterized in that, The target molecule is HCV-RNA.

Images