Compositions, kits and methods for human cytomegalovirus detection

Abstract

The application provides a composition, a kit and a method for human cytomegalovirus detection, and relates to the technical field of molecular diagnosis.The composition comprises at least one set of human cytomegalovirus detection primer probe groups and multiplex DNA polymerase, wherein the primer probe groups comprise oligonucleotide upstream external primers, downstream external primers, upstream internal primers, downstream internal primers, upstream loop primers and downstream loop primers; the probe is a neck ring structure probe, and the multiplex DNA polymerase comprises strand displacement DNA polymerase and DNA polymerase with 5' end to 3' end exonuclease activity.The composition for human cytomegalovirus detection alleviates the technical problems that the LAMP amplification scheme in the prior art is poor in specificity and cannot realize single-tube multiplex detection.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics, and in particular to a composition, kit, and method for detecting human cytomegalovirus. Background Technology

[0002] Human cytomegalovirus (HCMV) is the fifth member of the human herpesvirus family. It can cause a wide range of clinical syndromes. Infected individuals with normal immune function usually have no clinical manifestations, while HCMV infection in immunocompromised patients may cause severe symptoms and even endanger their lives.

[0003] The transmission routes of HCMV include (1) vertical transmission from mother to child: HCMV infection can occur at any stage of pregnancy. Most patients are asymptomatic and are classified as primary infection, secondary infection, and recurrent infection. Some patients may be reinfected with different HCMV virus strains. HCMV can be transmitted through the placenta, vaginal secretions, blood, saliva, and breast milk. HCMV can be excreted through the urogenital tract, so newborns can be infected through contact with vaginal secretions or blood. (2) horizontal transmission: Saliva is a common medium for horizontal transmission. HCMV can be transmitted through close contact between people. (3) iatrogenic transmission: HCMV can be transmitted through iatrogenic transmission methods such as blood transfusion, surgery, and organ transplantation.

[0004] Primary HCMV infection is often asymptomatic or presents with minimal clinical symptoms. Mononucleosis is the most common manifestation of HCMV in immunocompetent patients, characterized by fever, rash, and leukocytosis. In typical EBV mononucleosis, the main causative factors include the presence of pharyngeal secretions and heterophile antibodies, features typically absent in HCMV mononucleosis. Other manifestations of HCMV infection include anemia, abnormal liver function, thrombocytopenia, positive rheumatoid factor, and positive antinuclear antibodies. Organ involvement is uncommon in immunocompetent hosts. However, HCMV is a potential pathogen in immunosuppressed patients, such as those with HIV, solid organ transplant recipients, and bone marrow transplant recipients. This may be associated with specific organ diseases such as hepatitis, pneumonia, and colitis. HCMV is more aggressive in immunocompromised patients. Specific diseases include: CMV hepatitis, which can lead to fulminant hepatic failure; CMV retinitis; CMV esophagitis; CMV colitis; CMV pneumonia; polyradiculopathy; transverse myelitis; and subacute encephalitis.

[0005] Currently, the main methods for detecting human cytomegalovirus (CMV) include direct smear method, virus isolation and culture method, serological detection method, and nucleic acid detection method. While the direct smear method is intuitive, rapid, and inexpensive, its positive detection rate is low, and the results are easily affected by subjective and objective factors, leading to false results. Virus isolation and culture method is cumbersome, requires high precision, and is time-consuming, making it unsuitable for processing large volumes of clinical samples and for early diagnosis. Serological methods can only detect IgM in the early stages of herpesvirus infection, and often show negative results during the incubation period and recurrent infections, failing to reflect the true extent of infection. PCR technology amplifies the target virus in large quantities in vitro and combines it with molecular biology techniques for detection. Most current nucleic acid detection technologies are based on PCR, with quantitative real-time PCR (qPCR) being the most common. With the increasing demands of biotechnology in clinical and field testing, isothermal amplification techniques for nucleic acids have emerged as alternatives to PCR. Among these, loop-mediated isothermal amplification (LAMP) is a novel technique that utilizes DNA polymerases with strand displacement properties to catalyze the amplification of recognized nucleic acid sequences. It completes nucleic acid amplification under isothermal conditions and boasts high sensitivity, making it widely applicable in disease diagnosis, prognosis assessment, and prognostic monitoring. However, LAMP suffers from poor specificity and is prone to aerosol contamination. Furthermore, dye-based LAMP amplification protocols cannot achieve multiplex detection in a single tube. Therefore, improving the detection of human cytomegalovirus using LAMP technology is a problem that needs to be addressed.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a composition for detecting human cytomegalovirus, which alleviates the technical problem of poor specificity of the LAMP amplification protocol in the prior art, which makes it impossible to achieve multiplex detection in a single tube.

[0008] A second objective of this invention is to provide a kit for detecting human cytomegalovirus.

[0009] A third objective of this invention is to provide a method for detecting human cytomegalovirus for non-diagnostic and non-therapeutic purposes.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] According to one aspect of the present invention, the present invention provides a composition for detecting human cytomegalovirus, comprising: at least one set of human cytomegalovirus detection primers and probes and a multiplex DNA polymerase;

[0012] The human cytomegalovirus detection primer and probe set includes an upstream external primer of oligonucleotide, a downstream external primer of oligonucleotide, an upstream internal primer of oligonucleotide, a downstream internal primer of oligonucleotide, an upstream loop primer of oligonucleotide, a downstream loop primer of oligonucleotide, and an oligonucleotide fluorescent probe.

[0013] The oligonucleotide fluorescent probe has a stem-loop structure. The 5' end of the oligonucleotide fluorescent probe and the 3' end of the oligonucleotide fluorescent probe are complementary to each other, and the remaining bases are complementary to the sequence in the detection target. One end of the oligonucleotide fluorescent probe is modified with a luminescent group, and the other end is modified with a quenching group.

[0014] The multiplex DNA polymerase includes a strand displacement DNA polymerase and a DNA polymerase with 5' to 3' exonucleation activity.

[0015] Preferably, the oligonucleotide fluorescent probe has 10 bases starting at the 5' end and 10 bases starting at the 3' end that are complementary;

[0016] Preferably, the oligonucleotide fluorescent probe is complementary to the detection target at a position between the F1c region and the B1 region.

[0017] Preferably, the luminescent group is selected from FAM, Alexa Fluor 488, ATTO488, TET, JOE, VIC, Alexa Fluor 532, ATTO532, HEX, ROX, Alexa Fluor 594, ATTO594, TAMRA, CY3, CY3.5, CY5, Alexa Fluor 647, ATTO647, CY5.5, OregonGreen™, CALRed™, Red640, TexasRed, Alexa Fluor700, or ATTO700;

[0018] Preferably, the quenching group is selected from Super Quencher 1, BHQ1, BHQ2, BHQ3, Dabcyl, or MGB;

[0019] Preferably, the oligonucleotide fluorescent probe is modified with a luminescent group at its 5' end and a quenching group at its 3' end.

[0020] Preferably, the multiplex DNA polymerase is a hot-start DNA polymerase;

[0021] Preferably, the strand displacement DNA polymerase includes Bst DNA polymerase;

[0022] Preferably, the DNA polymerase having 5' to 3' exonuclease activity includes Taq DNA polymerase.

[0023] Preferably, the composition further includes one or more of the following: buffering substances, dNTPs, system balancing agents, betaine, salt, BSA, glycerol, and enzymes to prevent contamination of amplification products;

[0024] Preferably, the detection target includes one or more of the following genes: UL97, UL55, UL123, UL54, and UL42, in the human cytomegalovirus detection composition.

[0025] Preferably, the detection target includes the UL123 gene and the UL54 gene of the human cytomegalovirus detection composition;

[0026] Preferably, the detection target of the human cytomegalovirus detection primer and probe set is the UL54 gene, including:

[0027] The nucleotide sequences are as follows: upstream external primer 54-F3 (Seq_1); downstream external primer 54-B3 (Seq_2); upstream internal primer 54-FIP (Seq_3); downstream internal primer 54-BIP (Seq_4); upstream loop primer 54-LF (Seq_5); downstream loop primer 54-LB (Seq_6); and oligonucleotide fluorescent probe 54-EP (Seq_7).

[0028] Preferably, the 5' end of the oligonucleotide fluorescent probe 54-EP is modified with FAM, and the 3' end is modified with MGB;

[0029] Preferably, the detection target of the human cytomegalovirus detection primer and probe set is the UL123 gene, including: an upstream external primer 123-F3 of the oligonucleotide as shown in Seq_8; a downstream external primer 123-B3 of the oligonucleotide as shown in Seq_9; an upstream internal primer 123-FIP of the oligonucleotide as shown in Seq_10; a downstream internal primer 123-BIP of the oligonucleotide as shown in Seq_11; an upstream loop primer 123-LF of the oligonucleotide as shown in Seq_12; a downstream loop primer 123-LB of the oligonucleotide as shown in Seq_13; and an oligonucleotide fluorescent probe 123-EP as shown in Seq_14.

[0030] Preferably, the 5' end of the oligonucleotide fluorescent probe 123-EP is modified with VIC, and the 3' end is modified with MGB.

[0031] According to another aspect of the present invention, a kit for detecting human cytomegalovirus is also provided, the kit comprising the above-described composition.

[0032] Preferably, the kit contains an amplification reaction solution, which includes a buffer, dNTPs, a system balancing agent, betaine, salt, BSA, glycerol, and an enzyme to prevent contamination of the amplification products.

[0033] Preferably, the amplification reaction solution includes at least one set of human cytomegalovirus detection primer and probe set, Tris-HCl buffer, dATP, dCTP, dUTP, dGTP, dTTP, hot-start Bst DNA polymerase, hot-start Taq DNA polymerase, UNG enzyme, EGTA, magnesium chloride, potassium chloride, betaine, ammonium sulfate, BSA, and glycerol.

[0034] Preferably, the kit further includes a viral lysis buffer comprising: Tris-HCl 10–20 mM, N-lauroyl sarcosinate sodium 0.3–0.5% w / v, Tween 20 0.3–0.5% v / v, sodium chloride 80–120 mM, EDTA 0.5–1 mM, and glycerol 5–10% v / v;

[0035] Preferably, the virus lysis buffer comprises: Tris-HCl 15mM, N-lauroyl sarcosinate sodium 0.5% w / v, Tween 20 0.5% v / v, sodium chloride 100mM, EDTA 1mM and glycerol 5% v / v;

[0036] Preferably, the kit further includes one or more of a positive control, a negative control, and a quality control.

[0037] According to another aspect of the present invention, the present invention also provides a method for detecting human cytomegalovirus for non-diagnostic and therapeutic purposes, comprising amplifying a sample to be tested using the above-described composition or the above-described kit.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The composition for human cytomegalovirus (CMV) detection provided by this invention employs a dual polymerase amplification system, comprising a strand displacement DNA polymerase and a DNA polymerase with 5' to 3' exonuclease activity. The composition contains at least one set of human CMV detection primers and probes and a multiplex DNA polymerase. The primer and probe set includes an upstream external primer, a downstream external primer, an upstream internal primer, a downstream internal primer, an upstream loop primer, and a downstream loop primer for LAMP amplification. The probes are neck-loop structure probes, which effectively reduce the formation of secondary structures between the fluorescent probe and other oligonucleotide primers in the system.

[0040] The composition for human cytomegalovirus (CMV) detection provided by this invention utilizes a dual polymerase amplification system (LAMP-Taq-PCR) to amplify the target fragment of CMV. The primers and strand displacement DNA polymerase in this composition amplify the detection target using LAMP technology. A DNA polymerase with exonuclease activity from the 5' to the 3' end hydrolyzes the probe, causing the fluorescence signal to increase with the increase of the target gene fragment, enabling real-time monitoring of the amplified product. Using this composition to amplify the target fragment of CMV exhibits high efficiency, high sensitivity, and high specificity, enabling rapid, highly sensitive, and highly specific detection of CMV within 30 minutes. Attached Figure Description

[0041] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 This is a schematic diagram illustrating the binding of the primers and probes to the target gene in this invention;

[0043] Figure 2 This is a schematic diagram of a stem-loop structured oligonucleotide fluorescent probe.

[0044] Figure 3 The amplification results of human cytomegalovirus using the kit from Example 1 are shown below.

[0045] Figure 4 The amplification results of human genomic DNA using the kit from Example 1 are shown below.

[0046] Figure 5 The amplification results of human herpesvirus type 6 using the kit in Example 1;

[0047] Figure 6 The amplification results of human cytomegalovirus using the kit in Comparative Example 1 are shown.

[0048] Figure 7 The amplification results of human genomic DNA using the kit in Comparative Example 1 are shown.

[0049] Figure 8 The amplification results of human herpesvirus type 6 using the kit in Comparative Example 1 are shown.

[0050] Figure 9 The amplification results of the kit in Example 2 at a human cytomegalovirus concentration of 1000 copies / mL are shown, where A is the UL54 target and B is the UL123 target.

[0051] Figure 10 The amplification results of the kit in Example 2 at a human cytomegalovirus concentration of 500 copies / mL are shown, where A is the UL54 target and B is the UL123 target.

[0052] Figure 11 The results are amplification results of the kit in Example 2 when the concentration of human cytomegalovirus is 250 copies / mL, where A is the UL54 target and B is the UL123 target. Detailed Implementation

[0053] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] It should be noted that, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions; all technical features and preferred features mentioned herein can be combined to form new technical solutions; and the components involved or their preferred components can be combined to form new technical solutions. In this invention, unless otherwise specified, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are both real numbers.

[0055] According to one aspect of the present invention, a composition for detecting human cytomegalovirus is provided, the composition comprising at least one set of human cytomegalovirus detection primers and probes and a multiplex DNA polymerase.

[0056] The human cytomegalovirus (CMV) detection primer and probe set includes an upstream external primer, a downstream external primer, an upstream internal primer, a downstream internal primer, an upstream loop primer, a downstream loop primer, and a fluorescent oligonucleotide probe. For details on the binding modes of the primers and probes to the target gene, please refer to [link to relevant documentation]. Figure 1 LAMP primers are designed to target six different regions of the target gene. Four types of primers are designed based on six different sites: the F3c, F2c, and Flc regions at the 3' end of the target gene, and the B1, B2, and B3 regions at the 5' end. Figure 1 As shown. The upstream inner primer of the oligonucleotide consists of the F2 and F1c regions. The F2 region is complementary to the F2c region at the 3' end of the target gene, and the F1C region has the same sequence as the Flc region at the 5' end of the target gene. The downstream inner primer of the oligonucleotide consists of the B1C and B2 regions. The B2 region is complementary to the B2c region at the 3' end of the target gene, and the B1c region has the same sequence as the Blc region at the 5' end of the target gene. The upstream outer primer of the oligonucleotide consists of the F3 region and is complementary to the F3c region of the target gene. The downstream outer primer of the oligonucleotide consists of the B3 region and is complementary to the B3c region of the target gene. The oligonucleotide loop primer is used to increase the efficiency of LAMP amplification.

[0057] The oligonucleotide fluorescent probe has a stem-loop structure; see [link to relevant documentation]. Figure 2 The oligonucleotide fluorescent probe has an external base sequence starting at the 5' end, consisting of 9-11 bases (e.g., 9, 10, or 11 bases). This external base sequence is complementary to the 9-11 bases starting at the 3' end (e.g., 9, 10, or 11 bases), forming a stem-loop structure. Preferably, the oligonucleotide fluorescent probe has complementary base pairs at the 5' and 3' ends. The stem-loop structure fully opens at temperatures greater than or equal to 65°C. Designing the oligonucleotide fluorescent probe with a stem-loop structure effectively reduces the formation of secondary structures between the fluorescent probe and other oligonucleotide primers in the system.

[0058] Except for the ligand sequence, the bases of the oligonucleotide fluorescent probe are complementary to the detection target. The complementary pairing of the oligonucleotide fluorescent probe and the detection target is preferably located between the F1c region and the B1 region.

[0059] Furthermore, one end of the oligonucleotide fluorescent probe is modified with a luminescent group, and the other end is modified with a quenching group. The luminescent group and the quenching group can be selected from well-known and mature groups in the art. The luminescent group can be, for example, but not limited to, FAM, Alexa Fluor 488, ATTO488, TET, JOE, VIC, Alexa Fluor 532, ATTO532, HEX, ROX, Alexa Fluor 594, ATTO594, TAMRA, CY3, CY3.5, CY5, Alexa Fluor 647, ATTO647, CY5.5, OregonGreen™, CALRed™, Red640, TexasRed, Alexa Fluor700, or ATTO700; the quenching group can be, for example, but not limited to, Super Quencher 1, BHQ1, BHQ2, BHQ3, Dabcyl, or MGB. The luminescent group is preferably modified at the 5' end of the oligonucleotide fluorescent probe, and the quenching group is preferably modified at the 3' end of the oligonucleotide fluorescent probe.

[0060] The multiple DNA polymerases in this composition include a strand displacement DNA polymerase and a DNA polymerase with 5' to 3' exonuclease activity. Both the strand displacement DNA polymerase and the DNA polymerase with 5' to 3' exonuclease activity are preferably hot-start enzymes, wherein the strand displacement DNA polymerase preferably includes Bst DNA polymerase, and the DNA polymerase with 5' to 3' exonuclease activity preferably includes Taq DNA polymerase.

[0061] The composition for human cytomegalovirus (CMV) detection provided by this invention utilizes a dual polymerase amplification system (LAMP-Taq-PCR) to amplify the target fragment of CMV. The primers and strand displacement DNA polymerase in this composition amplify the detection target using LAMP technology. A DNA polymerase with exonuclease activity from the 5' to the 3' end hydrolyzes the probe, causing the fluorescence signal to increase with the increase of the target gene fragment, enabling real-time monitoring of the amplified product. Using this composition to amplify the target fragment of CMV exhibits high efficiency, high sensitivity, and high specificity, enabling rapid, highly sensitive, and highly specific detection of CMV within 30 minutes.

[0062] In some optional embodiments, the composition further includes one or more of the following: a buffer substance, dNTPs, a system balancing agent, betaine, salt, BSA, glycerol, and an enzyme to prevent contamination of amplification products. The buffer substance preferably includes Tris-HCl; the dNTPs preferably contain dUTP; the system balancing agent preferably includes EGTA (ethylene glycol-bis-(2-aminoethyl ether)tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), arginine, sodium citrate, or sodium gluconate, which can dynamically adjust the concentration of divalent cations to improve the specificity of the amplification system; the salt preferably includes a salt that provides magnesium and potassium ions, such as, but not limited to, potassium chloride, magnesium chloride, or magnesium sulfate, and the salt preferably also includes ammonium sulfate; the enzyme to prevent contamination of amplification products includes UNG enzymes and / or UDG enzymes.

[0063] In some alternative embodiments, the composition for human cytomegalovirus detection, in addition to the above-mentioned at least one set of human cytomegalovirus detection primers and probes and multiplex DNA polymerase, further includes dATP, dCTP, dUTP, dGTP, dTTP, UNG enzyme, EGTA, magnesium chloride, potassium chloride, betaine, ammonium sulfate, BSA, and glycerol.

[0064] In some optional embodiments, the detection target of the composition for human cytomegalovirus detection includes one or more of the UL97 gene, UL55 gene, UL123 gene, UL54 gene, and UL42 gene. It is understood that when the detection target of the composition targets only at least two genes, the composition contains at least two sets of human cytomegalovirus detection primer-probe sets. Preferably, the detection target of the composition includes the UL123 gene and the UL54 gene of the human cytomegalovirus detection composition.

[0065] In some optional embodiments, the detection target of the human cytomegalovirus detection primer-probe set is the UL54 gene, including: an upstream external primer 54-F3 of the oligonucleotide as shown in Seq_1; a downstream external primer 54-B3 of the oligonucleotide as shown in Seq_2; an upstream internal primer 54-FIP of the oligonucleotide as shown in Seq_3; a downstream internal primer 54-BIP of the oligonucleotide as shown in Seq_4; an upstream circular primer 54-LF of the oligonucleotide as shown in Seq_5; a downstream circular primer 54-LB of the oligonucleotide as shown in Seq_6; and an oligonucleotide fluorescent probe 54-EP as shown in Seq_7. The oligonucleotide fluorescent probe 54-EP is preferably modified with FAM at the 5' end and MGB at the 3' end.

[0066] In some optional embodiments, the detection target of the human cytomegalovirus detection primer-probe set is the UL123 gene, including: an upstream external primer 123-F3 with the nucleotide sequence shown in Seq_8; a downstream external primer 123-B3 with the nucleotide sequence shown in Seq_9; an upstream internal primer 123-FIP with the nucleotide sequence shown in Seq_10; a downstream internal primer 123-BIP with the nucleotide sequence shown in Seq_11; an upstream loop primer 123-LF with the nucleotide sequence shown in Seq_12; a downstream loop primer 123-LB with the nucleotide sequence shown in Seq_13; and an oligonucleotide fluorescent probe 123-EP with the nucleotide sequence shown in Seq_14. Preferably, the oligonucleotide fluorescent probe 123-EP is modified with VIC at its 5' end and MGB at its 3' end.

[0067] According to another aspect of the present invention, a kit for detecting human cytomegalovirus is also provided, the kit comprising the above-described composition.

[0068] In some alternative embodiments, the kit includes an amplification reaction solution comprising a buffer, dNTPs, a system balancing agent, betaine, salt, BSA, glycerol, and an enzyme to prevent contamination of the amplification products.

[0069] In some optional embodiments, the amplification reaction solution includes at least one set of human cytomegalovirus (CMV) detection primers and probes, as well as Tris-HCl buffer, dATP, dCTP, dUTP, dGTP, dTTP, hot-start Bst DNA polymerase, hot-start Taq DNA polymerase, UNG enzyme, EGTA, magnesium chloride, potassium chloride, betaine, ammonium sulfate, BSA, and glycerol. This amplification reaction solution, through specific optimization and upgrades, enables multiplex amplification detection using a two-primer system, improving the kit's detection rate for CMV. The presence of dUTP and temperature-sensitive uracil glycosylase (UNG) in the amplification reaction solution effectively reduces the risk of aerosol contamination.

[0070] The preferred concentrations of the above components are as follows:

[0071] The preferred concentration of the Tris-HCl buffer is 10 to 50 mM, for example, but not limited to 10, 15, 20, 25, 30, 35, 40, 45 or 50 mM, and more preferably 20 mM.

[0072] The concentration of dATP is preferably 0.1 to 1 mM, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM, more preferably 0.5 mM. The concentration of dCTP is preferably 0.1 to 1 mM, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM, more preferably 0.5 mM. The concentration of dUTP is preferably 0.1 to 1 mM, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM, more preferably 0.2 mM. The concentration of dGTP is preferably 0.1 to 1 mM, and can be, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM, more preferably 0.5 mM. The concentration of dTTP is preferably 0.1 to 1 mM, and can be, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM, more preferably 0.4 mM.

[0073] The concentration of hot-start Taq DNA polymerase is preferably 1–5 U / reaction, for example, but not limited to 1, 2, 3, 4, or 5 U / reaction, more preferably 5 U / reaction. The concentration of hot-start Bst DNA polymerase is preferably 2–10 U / reaction, for example, but not limited to 2, 3, 4, 5, 6, 7, 8, 9, or 10 U / reaction, more preferably 6 U / reaction. The concentration of UNG enzyme is preferably 0.2–1 U / reaction, for example, but not limited to 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 U / reaction, more preferably 0.5 U / reaction.

[0074] The concentration of EGTA is preferably 1–10 mM, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM, more preferably 3 mM. The concentration of magnesium chloride is 2–10 mM, for example, but not limited to 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM, more preferably 5 mM. The concentration of potassium chloride is preferably 10–100 mM, for example, but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM, more preferably 50 mM. The concentration of betaine is preferably 50–300 mM, for example, but not limited to 50, 100, 150, 200, 250, or 300 mM, more preferably 150 mM. The concentration of ammonium sulfate is preferably 5–50 mM, for example, but not limited to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM, more preferably 5 mM. The concentration of glycerol is preferably 1–15% v / v, for example, but not limited to 1, 3, 5, 7, 9, 10, 12, 14, or 15% v / v, more preferably 5% v / v. The concentration of BSA is preferably 0.5–20 mg / mL, for example, but not limited to 0.5, 1, 5, 10, 12, 15, 18, or 20 mg / mL, more preferably 10 mg / mL.

[0075] In the amplification reaction solution, the concentrations of primers and probes in the human cytomegalovirus detection primer and probe set are independently 0.1–1 μM, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μM. The primer concentration range refers to the concentration range of each primer in each primer and probe set, specifically the concentration range of the upstream external primer, downstream external primer, upstream internal primer, downstream internal primer, upstream loop primer, or downstream loop primer.

[0076] The preferred concentrations of primers and probes targeting the UL54 gene are as follows: upstream external primer 54-F3 0.4 μM, downstream external primer 54-B3 0.4 μM, upstream internal primer 54-FIP 0.3 μM, downstream internal primer 54-BIP 0.3 μM, upstream loop primer 54-LF 0.3 μM, downstream loop primer 54-LB 0.3 μM, and fluorescent probe 54-EP 0.6 μM.

[0077] The preferred concentrations of primers and probes targeting the UL123 gene are as follows: upstream external primer 123-F3 0.25 μM, downstream external primer 123-B3 0.25 μM, upstream internal primer 123-FIP 0.4 μM, downstream internal primer 123-BIP 0.4 μM, upstream loop primer 123-LF 0.3 μM, downstream loop primer 123-LB 0.3 μM, and fluorescent probe 123-EP 0.8 μM.

[0078] In some optional embodiments, the amplification reaction solution includes: 0.4 μM of upstream external primer 54-F3, 0.4 μM of downstream external primer 54-B3, 0.3 μM of upstream internal primer 54-FIP, 0.3 μM of downstream internal primer 54-BIP, 0.3 μM of upstream circular primer 54-LF, 0.3 μM of downstream circular primer 54-LB, 0.6 μM of oligonucleotide fluorescent probe 54-EP, 20 mM Tris-HCl, 0.5 mM dATP, 0.5 mM dCTP, 0.2 mM dUTP, 0.5 mM dGTP, 0.4 mM dTTP, and hot-start Taq polymerase. DNA polymerase 5U / reaction, hot-start BstDNA polymerase 6U / reaction, UNG enzyme 0.5U / reaction, EGTA 3mM, magnesium chloride 5mM, potassium chloride 50mM, betaine 150mM, ammonium sulfate 5mM, and glycerol 5% v / v.

[0079] In some optional embodiments, the amplification reaction solution includes: 0.4 μM of the upstream external primer 54-F3, 0.4 μM of the downstream external primer 54-B3, 0.3 μM of the upstream internal primer 54-FIP, 0.3 μM of the downstream internal primer 54-BIP, 0.3 μM of the upstream circular primer 54-LF, 0.3 μM of the downstream circular primer 54-LB, 0.6 μM of the oligonucleotide fluorescent probe 54-EP, 0.25 μM of the upstream external primer 123-F3, 0.25 μM of the downstream external primer 123-B3, 0.4 μM of the upstream internal primer 123-FIP, 0.4 μM of the downstream internal primer 123-BIP, and 0.4 μM of the upstream circular primer 123-LF. 0.3 μM, oligonucleotide downstream loop primer 123-LB 0.3 μM, oligonucleotide fluorescent probe 123-EP 0.8 μM, Tris-HCl 20 mM, dATP 0.5 mM, dCTP 0.5 mM, dUTP 0.2 mM, dGTP 0.5 mM, dTTP 0.4 mM, hot-start Taq DNA polymerase 5 U / reaction, hot-start Bst DNA polymerase 6 U / reaction, UNG enzyme 0.5 U / reaction, EGTA 3 mM, magnesium chloride 5 mM, potassium chloride 50 mM, betaine 150 mM, ammonium sulfate 5 mM, and glycerol 5% v / v.

[0080] In some optional embodiments, the kit for detecting human cytomegalovirus further includes a viral lysis buffer comprising: Tris-HCl 10–20 mM, for example, but not limited to, 10, 12, 15, 17, or 20 mM; N-lauroyl sarcosinate sodium 0.3–0.5% w / v, for example, but not limited to, 0.3, 0.4, or 0.5% w / v; and Tween 20. 0.3–0.5% v / v, for example, but not limited to 0.3, 0.4, or 0.5% v / v; sodium chloride 80–120 mM, for example, but not limited to 80, 90, 100, 110, or 120 mM; EDTA 0.5–1 mM, for example, but not limited to 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mM; and glycerol 5–10% v / v, for example, but not limited to 5, 6, 7, 8, 9, or 10% v / v. This viral lysis buffer is compatible with the LAMP method. Human cytomegalovirus, after being lysed with the viral lysis buffer, can be directly added to the LAMP amplification system for direct amplification without interference from the viral lysis buffer.

[0081] In some alternative embodiments, the viral lysis buffer comprises Tris-HCl 15 mM, N-lauroyl sarcosinate sodium 0.5% w / v, Tween 20 0.5% v / v, sodium chloride 100 mM, EDTA 1 mM, and glycerol 5% v / v.

[0082] In some alternative embodiments, the kit may further include one or more of a positive control, a negative control, and a quality control, preferably including a quality control.

[0083] According to another aspect of the present invention, a method for detecting human cytomegalovirus (CMV) for non-diagnostic and non-therapeutic purposes is provided, comprising amplifying a sample to be tested using the above-described composition or kit. Since the amplification system of the composition or kit provided by the present invention contains a probe, the method provided by the present invention can directly determine whether a sample to be tested contains CMV through fluorescence. The detection results can be determined according to general common knowledge in the art, for example, based on amplification curves, Ct values, or by constructing standard curves in a qualitative, semi-quantitative, or quantitative manner; the present invention does not limit this.

[0084] The sample to be tested is preferably pretreated with the above-mentioned viral lysis buffer, and then amplified using the sample processing solution as a template. The pretreatment preferably includes mixing the viral lysis buffer with the sample to be tested and incubating at at least 60°C for at least 5 minutes, more preferably at 65°C for 5 minutes.

[0085] In some preferred embodiments, the amplification program is preferably: (a) 25°C for 5 min; (b) 60°C for 20 min; (c) 95°C for 10 s, 70°C for 30 s, with signal detection, for 10 cycles.

[0086] The technical solution and beneficial effects of the present invention will be further described below with reference to preferred embodiments.

[0087] The sequence information in the following implementation is shown in Table 1:

[0088] Table 1. Sequence information for the human cytomegalovirus nucleic acid detection kit

[0089]

[0090]

[0091] Example 1

[0092] This embodiment provides a LAMP-Taq-PCR human cytomegalovirus detection kit, comprising: viral lysis buffer and LAMP-Taq-PCR amplification reaction solution. The formulation of the viral lysis buffer is shown in Table 1, the formulation of the LAMP-Taq-PCR amplification reaction solution is shown in Table 2, and the sequence information of the primers and probes in the LAMP-Taq-PCR amplification reaction solution is shown in Table 1.

[0093] Table 2. Components and concentrations of virus lysate

[0094] Components Component concentration Tris-HCl 15mM Sodium N-lauroyl sarcosinate 0.5% (w / v) Twain 20 0.5% (v / v) Sodium chloride 100mM EDTA 1mM glycerin 5% (v / v)

[0095] Table 3. Components and concentrations of the LAMP-Taq-PCR amplification reaction solution in Example 1

[0096]

[0097]

[0098] Example 2

[0099] This embodiment provides a LAMP-Taq-PCR human cytomegalovirus detection kit. The detection targets of this kit are the UL123 and UL54 genes, and it includes: viral lysis buffer and LAMP-Taq-PCR amplification reaction solution. The formulation of the viral lysis buffer is shown in Table 1, the formulation of the LAMP-Taq-PCR amplification reaction solution is shown in Table 4, and the sequence information of the primers and probes in the LAMP-Taq-PCR amplification reaction solution is shown in Table 1.

[0100] Table 4. Components and concentrations of the LAMP-Taq-PCR amplification reaction solution in Example 2

[0101]

[0102]

[0103] Comparative Example 1

[0104] This comparative example provides a LAMP human cytomegalovirus detection kit, including viral lysis buffer and conventional LAMP amplification reaction solution. The formulation of the viral lysis buffer is shown in Table 1, and the formulation of the LAMP amplification reaction solution is shown in Table 5. The sequence information of the primers and probes in the amplification reaction solution is shown in Table 1. The main difference between the comparative example LAMP reaction solution and the LAMP-Taq-PCR amplification reaction solution in Example 1 is that it does not contain Taq DNA polymerase and stem-loop probe 54-EP, and it also contains Syto82.

[0105] Table 5. Composition and concentration of each component of the conventional LAMP amplification reaction solution in Comparative Example 1

[0106]

[0107]

[0108] Example 1

[0109] Take 10 μL each of human cytomegalovirus standard, human genomic DNA extract and human herpesvirus type 6 standard (WHO), mix them separately with 10 μL of viral nucleic acid lysis buffer, incubate at 65 degrees Celsius for 5 minutes, and set aside.

[0110] Take 15 μL of the above sample processing solution and mix it with 20 μL of the LAMP-Taq-PCR amplification reaction solution from Example 1 and the conventional LAMP amplification reaction solution from Comparative Example 1. Then, perform nucleic acid detection according to the LAMP-Taq-PCR amplification program in Table 6 and the conventional LAMP amplification program in Table 7, respectively, and compare their amplification specificity. The structure is as follows: Figures 3-8 As shown in the figure. The results showed that for human cytomegalovirus nucleic acid, both LAMP-Taq-PCR and conventional LAMP amplification protocols could detect signals normally. For human genomic DNA extract, neither LAMP-Taq-PCR nor conventional LAMP amplification protocols showed non-specific amplification signals. For human herpesvirus type 6 nucleic acid, the LAMP-Taq-PCR protocol showed no non-specific amplification signals, while the conventional LAMP amplification protocol showed non-specific amplification signals after 15 minutes of reaction.

[0111] Table 6. Amplification Procedure for LAMP-Taq-PCR Detection Kit for Human Cytomegalovirus Nucleic Acid

[0112]

[0113] Table 7. Amplification Procedure for LAMP Assay Kit for Human Cytomegalovirus Nucleic Acid Detection

[0114] 25 degrees 5min 1 60 degrees 1 min (√) 25

[0115] In Tables 6 and 7, “√” indicates that signal detection is performed in this program segment.

[0116] Example 2

[0117] Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus type 6, human herpesvirus type 7, human herpesvirus type 8, human immunodeficiency virus (HIV), hepatitis B virus (HBV), influenza A virus, and adenovirus (HAdV) standards were selected. Virus lysis was performed using the virus lysis buffer from Example 2, and the mixture was incubated at 65 degrees Celsius for 5 minutes. Then, 15 μL of the virus lysis buffer was mixed with 20 μL of the LAMP-Taq-PCR amplification reaction solution from Example 2, and amplification and detection were performed according to the procedure in Table 6. The results are shown in Table 8. The results indicate that the kit from Example 2 detected no non-specific signals for the above virus standards.

[0118] Table 8. Specificity Analytical Standards

[0119]

[0120]

[0121] In the test results, "+" indicates a positive result, and "-" indicates a negative result.

[0122] Example 3

[0123] Sensitivity analysis: Human cytomegalovirus nucleic acid standard (GBW(E)090676; Guangzhou Bondsheng Biotechnology Co., Ltd.) was diluted to 1000 copies / mL, 500 copies / mL, and 250 copies / mL, respectively. The sensitivity of these concentrations was then evaluated using the human cytomegalovirus nucleic acid detection kit from Example 2. The results are as follows: Figures 9-11 As shown, the results indicate that when the concentration of human giant cell standard is 250 copies / mL, there is still a positive signal detection, indicating that the sensitivity of the kit of the present invention is lower than 250 copies / mL.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for human cytomegalovirus detection, characterized by, The application relates to a human cytomegalovirus detection primer probe set and a multiplex DNA polymerase. The human cytomegalovirus detection primer probe set comprises an oligonucleotide upstream external primer, an oligonucleotide downstream external primer, an oligonucleotide upstream internal primer, an oligonucleotide downstream internal primer, an oligonucleotide upstream loop primer, an oligonucleotide downstream loop primer and an oligonucleotide fluorescent probe. The oligonucleotide fluorescent probe is a stem-loop structure, 9-11 bases at the 5' end and 9-11 bases at the 3' end of the oligonucleotide fluorescent probe are complementary to each other, and the remaining bases are complementary to the sequence in the detection target; one end of the oligonucleotide fluorescent probe is modified with a luminescent group, and the other end is modified with a quenching group. The multiplex DNA polymerase is Bst DNA polymerase and Taq DNA polymerase. 10 bases at the 5' end and 10 bases at the 3' end of the oligonucleotide fluorescent probe are complementary to each other; the position of the oligonucleotide fluorescent probe which is complementary to the detection target is between the F1c region and the B1 region. The detection target of the two groups of human cytomegalovirus detection primer probe sets is the UL123 gene and the UL54 gene. The human cytomegalovirus detection primer probe set with the UL54 gene as the detection target comprises an oligonucleotide upstream external primer 54-F3 with the nucleotide sequence shown in SEQ ID NO. 1, an oligonucleotide downstream external primer 54-B3 with the nucleotide sequence shown in SEQ ID NO. 2, an oligonucleotide upstream internal primer 54-FIP with the nucleotide sequence shown in SEQ ID NO. 3, an oligonucleotide downstream internal primer 54-BIP with the nucleotide sequence shown in SEQ ID NO. 4, an oligonucleotide upstream loop primer 54-LF with the nucleotide sequence shown in SEQ ID NO. 5, an oligonucleotide downstream loop primer 54-LB with the nucleotide sequence shown in SEQ ID NO. 6 and an oligonucleotide fluorescent probe 54-EP with the nucleotide sequence shown in SEQ ID NO.

7. The human cytomegalovirus detection primer probe set with the UL123 gene as the detection target comprises an oligonucleotide upstream external primer 123-F3 with the nucleotide sequence shown in SEQ ID NO. 8, an oligonucleotide downstream external primer 123-B3 with the nucleotide sequence shown in SEQ ID NO. 9, an oligonucleotide upstream internal primer 123-FIP with the nucleotide sequence shown in SEQ ID NO. 10, an oligonucleotide downstream internal primer 123-BIP with the nucleotide sequence shown in SEQ ID NO. 11, an oligonucleotide upstream loop primer 123-LF with the nucleotide sequence shown in SEQ ID NO. 12, an oligonucleotide downstream loop primer 123-LB with the nucleotide sequence shown in SEQ ID NO. 13 and an oligonucleotide fluorescent probe 123-EP with the nucleotide sequence shown in SEQ ID NO.

14. ​ 2. The composition according to claim 1, characterized in that, the luminescent group is selected from FAM, Alexa Fluor 488, ATTO 488, TET, JOE, VIC, Alexa Fluor 532, ATTO 532, HEX, ROX, Alexa Fluor 594, ATTO 594, TAMRA, CY3, CY3.5, CY5, Alexa Fluor 647, ATTO 647, CY5.5, Oregon GreenTM, CAL RedTM, Red 640, Texas Red, Alexa Fluor 700 or ATTO 700; the quenching group is selected from Super Quencher 1, BHQ1, BHQ2, BHQ3, Dabcyl or MGB; the 5' end of the oligonucleotide fluorescent probe is modified with the luminescent group and the 3' end is modified with the quenching group.

3. The composition of claim 1, wherein, the composition further comprises one or more of a buffer substance, dNTPs, a system balancing agent, betaine, a salt, BSA, glycerol and an enzyme for preventing contamination of amplification products.

4. The composition of claim 3, wherein, the buffer substance comprises Tris-HCl; the dNTPs comprise dUTP; the system balancing agent comprises EGTA, EDTA, arginine, sodium citrate or sodium malate; the salt comprises ammonium sulfate and a salt providing magnesium ions and potassium ions; the enzyme for preventing contamination of amplification products comprises UNG enzyme and / or UDG enzyme.

5. The composition of claim 1, wherein the composition further comprises dATP, dCTP, dUTP, dGTP, dTTP, UNG enzyme, EGTA, magnesium chloride, potassium chloride, betaine, ammonium sulfate, BSA and glycerol.

6. The composition according to any one of claims 1 to 5, characterized in that, the 5' end of the oligonucleotide fluorescent probe 54-EP is modified with FAM and the 3' end is modified with MGB; the 5' end of the oligonucleotide fluorescent probe 123-EP is modified with VIC and the 3' end is modified with MGB.

7. A kit for the detection of human cytomegalovirus, characterized in that, comprising the composition of any one of claims 1-6.

8. The kit of claim 7, wherein the kit comprises an amplification reaction solution comprising a buffer substance, dNTPs, a system balancing agent, betaine, a salt, BSA, glycerol and an enzyme for preventing contamination of amplification products.

9. The kit of claim 8, wherein the amplification reaction system of the kit comprises two groups of human cytomegalovirus detection primer probes, Tris-HCl buffer, dATP, dCTP, dUTP, dGTP, dTTP, hot-start Bst DNA polymerase, hot-start Taq DNA polymerase, UNG enzyme, EGTA, magnesium chloride, potassium chloride, betaine, ammonium sulfate, BSA and glycerol; the concentration of the Tris-HCl buffer is 10-50 mM; the concentrations of dATP, dCTP, dUTP, dGTP and dTTP are independently 0.1-1 mM; the concentration of the hot-start Taq DNA polymerase is 1-5 U / reaction; the concentration of the hot-start Bst DNA polymerase is 2-10 U / reaction; the concentration of the UNG enzyme is 0.2-1 U / reaction; the concentration of the EGTA is 1-10 mM; the concentration of the magnesium chloride is 2-10 mM; the concentration of the potassium chloride is 10-100 mM; The concentration of betaine is 50-300 mM; The concentration of ammonium sulfate is 5-50 mM; The concentration of glycerol is 1-15 %v / v; The concentration of BSA is 0.5-20 mg / mL; The concentration of the primers and probes in the primer-probe set for human cytomegalovirus detection is independently 0.1-1 μM.

10. The kit of claim 9, wherein The concentration of Tris-HCl buffer is 20 mM; The concentration of dATP, dCTP and dGTP is 0.5 mM; The concentration of dUTP is 0.2 mM; The concentration of dTTP is 0.4 mM; The concentration of hot-start Taq DNA polymerase is 5 U / reaction; The concentration of hot-start Bst DNA polymerase is 6 U / reaction; The concentration of UNG enzyme is 0.5 U / reaction; The concentration of EGTA is 3 mM; The concentration of magnesium chloride is 5 mM; The concentration of potassium chloride is 50 mM; The concentration of betaine is 150 mM; The concentration of ammonium sulfate is 5 mM; The concentration of glycerol is 5 %v / v; The concentration of BSA is 10 mg / mL.

11. The kit of claim 9, wherein The amplification reaction system comprises: 0.4 μM of oligonucleotide upstream external primer 54-F3, 0.4 μM of oligonucleotide downstream external primer 54-B3, 0.3 μM of oligonucleotide upstream internal primer 54-FIP, 0.3 μM of oligonucleotide downstream internal primer 54-BIP, 0.3 μM of oligonucleotide upstream loop primer 54-LF, 0.3 μM of oligonucleotide downstream loop primer 54-LB, 0.6 μM of oligonucleotide fluorescent probe 54-EP, 20 mM of Tris-HCl, 0.5 mM of dATP, 0.5 mM of dCTP, 0.2 mM of dUTP, 0.5 mM of dGTP, 0.4 mM of dTTP, 5 U / reaction of hot-start Taq DNA polymerase, 6 U / reaction of hot-start Bst DNA polymerase, 0.5 U / reaction of UNG enzyme, 3 mM of EGTA, 5 mM of magnesium chloride, 50 mM of potassium chloride, 150 mM of betaine, 5 mM of ammonium sulfate and 5 %v / v of glycerol.

12. The kit of claim 9, wherein The amplification reaction system further comprises: 0.25 μM of oligonucleotide upstream external primer 123-F3, 0.25 μM of oligonucleotide downstream external primer 123-B3, 0.4 μM of oligonucleotide upstream internal primer 123-FIP, 0.4 μM of oligonucleotide downstream internal primer 123-BIP, 0.3 μM of oligonucleotide upstream loop primer 123-LF, 0.3 μM of oligonucleotide downstream loop primer 123-LB, 0.8 μM of oligonucleotide fluorescent probe 123-EP.

13. The kit of claim 7, wherein Also included is a viral lysis solution comprising: Tris-HCl 10-20 mM, N-lauroylsarcosine sodium 0.3-0.5% w / v, Tween 20 0.3-0.5% v / v, sodium chloride 80-120 mM, EDTA 0.5-1 mM, and glycerol 5-10% v / v.

14. The kit of claim 13, wherein The viral lysis solution comprises: Tris-HCl 15 mM, N-lauroylsarcosine sodium 0.5% w / v, Tween 20 0.5% v / v, sodium chloride 100 mM, EDTA 1 mM, and glycerol 5% v / v. The kit further comprises one or more of a positive control, a negative control, and a quality control.

15. A method for detecting human cytomegalovirus for non-diagnostic and therapeutic purposes, characterized in that, The method comprises amplifying a sample to be tested using the composition of any one of claims 1-6, or the kit of any one of claims 7-14.

16. The method of claim 15, wherein, The method comprises pre-treating the sample to be tested using the viral lysis solution of claim 13, and then amplifying using the sample treatment solution as a template. The pre-treatment comprises mixing the viral lysis solution with the sample to be tested, and incubating at least 5 min at at least 60°C. The amplification procedure is: (a) 25°C for 5 min; (b) 60°C for 20 min; (c) 95°C for 10 s, 70°C for 30 s, and signal detection, 10 cycles.

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