CN115852046B

CN115852046B - Composition, kit, method and application for detecting and typing respiratory viruses

Info

Publication number: CN115852046B
Application number: CN202211189723.1A
Authority: CN
Inventors: 万旺; 刘璇; 陈晓亮; 任小梅; 刘佳; 戴立忠
Original assignee: Sansure Biotech Inc
Current assignee: Sansure Biotech Inc
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2024-01-30
Anticipated expiration: 2042-09-28
Also published as: CN115852046A

Abstract

The invention belongs to the field of molecular biology detection, and particularly relates to a composition for detecting and typing respiratory viruses, and more particularly relates to a composition, a kit, a method and application for detecting PIV-1, PIV-3 and HMPV and wolf tooth viruses. The invention provides a composition, which mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting different sites on different pathogens, thereby simultaneously realizing detection and typing of four viruses in a single-tube reaction system. So that different pathogens can be treated differently, thereby making the treatment and prevention more efficient. Meanwhile, the sensitivity of the composition can reach 200 copies/mL at the highest, and the detection is more accurate.

Description

Composition, kit, method and application for detecting and typing respiratory viruses

Technical Field

The invention belongs to the field of molecular biology detection, and particularly relates to a composition for detecting and typing respiratory viruses, and more particularly relates to a composition, a kit, a method and application for detecting PIV-1, PIV-3 and HMPV and wolf tooth viruses.

Background

Parainfluenza virus (PIV) is a polymorphic, enveloped, single-stranded negative-strand RNA virus of the Paramyxoviridae genus, divided into 4 subtypes, PIV-1 and PIV-3 belonging to the genus respiratory virus, and PIV-2 and PIV-4 belonging to the genus mumps virus. The virus diameter is 125-250nm, and the envelope is composed of lipid and glycoprotein. There are two types of glycoproteins: one is hemagglutinin-neuraminidase protein (NH) with hemagglutination activity and neuraminidase activity; the other is fusion protein (F protein) with cell fusion promoting effect and hemolysis property. The pathogenesis of PIV-induced diseases is mediated by viral replication and host immune responses. High levels of viral replication may cause alterations in airway epithelial cells, which are found in PIV infected patients to be associated with increased mucus secretion. Like other respiratory viral infections, host immune responses-innate immunity, antibody responses, T cell responses are also associated. PIV initially infects the pseudolaminar mucous airway epithelium of the nasal cavity and then passes through the oropharynx and into the airway. Disease severity is also related to the site of infection of PIV, with mild infection generally being limited to the upper respiratory tract and severe infection of PIV generally invading the lower respiratory tract, resulting in a more intense immune response. Whereas viremia only occurs in immunocompromised patients.

Over time, the incidence of the four subtypes varied, but three retrospective studies all showed that PIV-3 infection was the subtype that was clinically most susceptible to infection. The four subtypes show obvious seasonality, PIV-1 infection is popular once in two years, and 9-12 months of odd years are obviously increased; whereas the detonation of PIV-3 occurs at 4-6 months per year. PIV-3 is active in the non-epidemic years, and occurs in 10-12 months in addition to the spring popularity; similar to PIV-3, PIV-2 occurs annually, but on a smaller scale; PIV-4 is less isolated and detected, so it is difficult to derive epidemiological data.

Human Metapneumovirus (HMPV) is popular worldwide and has seasonal distribution characteristics, the detected peaks mainly appear in spring and winter, and the annual inpatient rate caused by infection is the same as that caused by influenza virus, parainfluenza virus types 1, 2 and 3. A recent study indicated that the seasonal peak of HMPV occurs after 3-4 months, i.e. the seasonal peaks of respiratory syncytial virus and influenza virus infection; another study indicated that HMPV infection seasons overlapped with RSV infection seasons; this indicates that the HMPV detection rate is not the same as the detection peak in each region. Many studies have reported the co-infection of HMPV with other respiratory pathogens (RSV, bocavirus, rhinovirus, parainfluenza virus, coronavirus, influenza a virus, influenza b virus).

Hendra virus and nipah virus belong to the family paramyxoviridae, henipavirus genus (henipavirus), and are known to infect humans and induce fatal disease; however, other related henipa viruses are also found in bats, rodents and shrews. A new animal-derived henipav that infects humans was found in a sample of throat swabs from a patient by high throughput sequencing and virus isolation and was designated as the wolf tooth virus (Langya henipavirus, layV). The clinical symptoms of 26 patients found were fever (100%), hypodynamia (54%), cough (50%), anorexia (50%), myalgia (46%), nausea (38%), headache (35%), vomiting (35%), with thrombocytopenia (35%), leukopenia (54%), impaired liver function (35%), impaired kidney function (8%), and the like.

The clinical manifestations of the 4 virus patients are similar, and the main symptoms are fever, cough, expectoration and wheezing. In children under 5 years old, PIVs infection is one of the major causes of community-acquired pneumonia in hospitalized children, being the second largest viral pathogen next to Respiratory Syncytial Virus (RSVs), far beyond influenza virus. PIVs are commonly infected, especially in lower respiratory tract infections, and PIVs often have combined infections with other pathogens. Epidemiological studies report that PIVs, like influenza, are more susceptible to streptococcus pneumoniae infection and can result in exacerbation of the infection. Etiology monitoring studies of lower respiratory tract infections in children suggest that PIVs can also cause exacerbations in combination with other viral infections.

Thus, there is a need in the art for a product that can simultaneously identify and type these respiratory viruses in order to establish patient causative factors for symptomatic treatment and reduce the risk of complications.

Disclosure of Invention

In view of this, in a first aspect, the present invention provides a composition for detecting and typing respiratory viruses, comprising:

upstream and downstream primers and probes for detecting PIV-1 shown in SEQ ID NO. 1-3;

upstream and downstream primers and probes for detecting PIV-3 shown in SEQ ID NO. 4-6;

the upstream and downstream primers and probes for detecting HMPV are shown as SEQ ID NO. 7-9; and

the upstream and downstream primers and probes for detecting the spike virus are shown as SEQ ID NO. 13-15.

The invention provides a composition, which mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting different sites on different pathogens, thereby simultaneously realizing detection and typing of four viruses in a single-tube reaction system. So that different pathogens can be treated differently, thereby making the treatment and prevention more efficient. Meanwhile, the sensitivity of the composition can reach 200 copies/mL at the highest, and the detection is more accurate.

Further, the composition further comprises: the upstream and downstream primers and probes for detecting HMPV are shown as SEQ ID NO. 10-12.

The use of the composition enables the simultaneous detection of two targets of HMPV, avoids the risk of missed detection that may exist due to mutation, reduces the probability of false negatives, and further increases the accuracy of detection.

Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.

As used herein, "distinct and non-interfering with each other" means that the fluorophores used by the probes in the composition to detect each target are not identical and do not interfere with each other's detection, i.e., can be detected using different channels. For example FAM, HEX, ROX and CY5 can be used, which groups do not have close absorbance values and can select different channels so as not to interfere with each other.

Further, in some embodiments, the compositions of the present invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to matched upstream and downstream primers and probes that detect a target.

The compositions of the invention can be combined in any combination to detect 5 targets. Those skilled in the art can combine the primers and probe pairs as necessary to detect which targets are the corresponding targets. These combinations are included in the present invention.

For example, any 4 pairs of the above 5 pairs of primers and probes may be included, any 3 pairs of the above 5 pairs of primers and probes may be included, any 2 pairs of the above 5 pairs of primers and probes may be included, or any 1 pair of the above 5 pairs of primers and probes may be included.

Further, the composition comprises: and detecting the upstream and downstream primers and probes of the internal standard.

In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is GAPDH.

In some specific embodiments, the compositions of the invention are used in fluorescent PCR.

In a specific embodiment, the fluorescent reporter group of the PIV-1 probe is FAM; the fluorescence reporter group of the PIV-3 probe is HEX; the fluorescence reporter group of HPMC is CY5; the fluorescent reporter group of the wolf virus is ROX.

Further, the 3' end of the probe also has a non-fluorescent quencher.

Further, the 3' -end of the probe also has a quenching group, such as SQ1, BQ1, SQ2 or BQ2.

In a specific embodiment, the 3' end of the probe is SQ1.

Further, the amount of the primer in the composition is 0.05 to 5. Mu.M; the amount of probe in the composition is 0.05 to 5. Mu.M.

In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.

In a particular embodiment, the ingredients of the composition of the invention are present in the same package.

Further, the components of the composition of the present invention are present in a mixed form.

In a second aspect, the invention provides the use of a composition of the invention as described above for the preparation of a kit for detecting and typing respiratory viruses, the pathogens being PIV-1, PIV-3, HMPV, and Langerhans' viruses.

In a third aspect, the present invention provides a kit for detecting and typing a respiratory virus, the kit comprising a composition of the invention as described above.

Further, the kit also comprises a negative quality control and a positive quality control.

In a specific embodiment, the negative quality control is DEPC H ₂ O, normal saline and internal standard gene pseudovirus. The positive quality control agent is at least one of HHV-7, HPV-11 and HPV-16 target gene fragment plasmid, RNA fragment and pseudovirus.

Further, the kit also comprises dNTP, PCR buffer solution and Mg ²⁺ At least one of them.

Still further, the kit further comprises: at least one of a nucleic acid releasing agent, a nucleic acid extracting reagent and a DNA polymerase.

Further, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTPs, a DNA polymerase, a PCR buffer solution and Mg ²⁺ At least one of them.

Further, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.

In a specific embodiment, the kit of the invention comprises:

PCR buffers, DNA polymerase, reverse transcriptase (RT enzyme), dntps, primers, probes, and metal cations required to catalyze DNA polymerase.

Common PCR buffer consists of Tris-HCl and MgCl ₂ Buffer systems such as KCl and Triton X-100. The total volume in a typical single PCR reaction tube is 20-200. Mu.l.

In a specific embodiment, the kit of the invention is compatible with digital PCR amplification systems, i.e., can be used directly on a digital PCR instrument for amplification.

In a fourth aspect, there is provided a method for detecting and typing a respiratory virus, the method comprising the steps of:

1) Extracting or releasing nucleic acid of a sample to be tested;

2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

3) The results were obtained and analyzed.

In the present invention, the sample for detection may be a pharyngeal swab, an oropharyngeal swab, vaginal secretion, blood, or the like, but is not limited thereto.

Further, the reaction conditions of the fluorescent quantitative PCR are as follows:

reverse transcription reaction at 50-60 deg.c for 5-20 min and 1 circulation; pre-denaturation at 90-95 deg.c for 1-5 min for 1 cycle; denaturation at 90-95 deg.c for 5-20 sec, annealing at 55-60 deg.c for 10-60 sec, 30-50 cycles, and collecting fluorescence.

In a specific embodiment, a method for detecting and typing respiratory viruses for non-diagnostic purposes is provided, the method comprising the steps of:

1) Extracting or releasing nucleic acid of a sample to be tested;

3) The results were obtained and analyzed.

As used herein, the term "non-diagnostic purpose" refers to information not intended to obtain whether an individual is infected with PIV-1, PIV-3, HMPV, and Langerhans' virus. For example, the method may be used to detect the presence or absence of the above-mentioned pathogens in test cultures in experiments aimed at scientific research.

Drawings

FIGS. 1-2 show the results of detection of compositions according to the invention (SEQ ID NOs 7-9 for detecting HMPV and SEQ ID NOs 7-12 for detecting HPMC, respectively);

FIGS. 3-6 are graphs showing the sensitivity of the compositions of the present invention;

FIG. 7 is a composition specificity of the present invention;

FIGS. 8 to 9 show the results of the comparative example composition of the present invention.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Example 1, primers and probes used in the present invention

TABLE 1

Wherein, the fluorescence reporter group of the PIV-1 probe is FAM; the fluorescence reporter group of the PIV-3 probe is HEX; the fluorescence report group of the HPMC probe is CY5; the fluorescent reporter group of the wolf tooth virus probe is ROX.

Example 2 method for detecting and typing human respiratory viruses

Reagent preparation:

and taking corresponding amounts of the PCR reaction liquid and the enzyme mixed liquid according to the quantity of the sample to be detected, the positive control and the negative control according to a proportion (38 mu L of the PCR reaction liquid/part+2 mu L of the enzyme mixed liquid/part), fully and uniformly mixing the PCR reaction liquid and the enzyme mixed liquid to form a PCR mixed liquid, and centrifuging at 2000rpm for 10s for later use.

Sample processing and sample addition

200. Mu.L of the sample to be tested, negative control, positive control were placed in a 1.5mL centrifuge tube, and nucleic acid extraction was performed using a nucleic acid extraction or purification reagent (S10015) from Sanxiang Biotechnology Co., ltd.

And (3) sucking 10 mu L of each of the treated sample, the negative control and the positive control, respectively adding the 10 mu L of each of the treated sample, the negative control and the positive control into a corresponding 0.2mL PCR reaction tube, adding 40 mu L of PCR mixed solution into each tube, and covering a tube cover.

PCR reaction system:

TABLE 2

PCR amplification

PCR amplification is carried out on PCR instruments such as an ABI7500 fluorescent quantitative PCR instrument, a Life Technologies Quant StudioTM fluorescent PCR instrument, a SLAN-96P full-automatic medical PCR analysis system and the like according to a certain temperature and time setting program. The preferred embodiment of the present invention is shown in Table 3.

TABLE 3 Table 3

* And (3) injection: for ABI7500 instrument reasons, it cannot be set to 30 seconds, and it can be set to 31 seconds or 32 seconds.

Interpretation of test results

If the sample FAM, HEX (VIC), ROX and CY5 channels have obvious S-shaped amplification curves and Ct value is less than or equal to 40, judging positive; if the sample FAM, HEX (VIC), ROX, CY5 channel has No amplification curve (No Ct) or Ct value > 40, then the sample is judged as negative. The details are shown in Table 4 below.

TABLE 4 Table 4

Example 3 detection results of test samples of the inventive composition

The primers and probes shown in example 1 were used to verify the pseudovirus samples according to the method of example 2, and the results showed that PIV-1, PIV-3, HMPV and Langerhans virus could be distinguished, and the detection results are shown in FIG. 1 (detection of HMPV containing only SEQ ID NOS: 7 to 9) and FIG. 2 (detection of HPMV containing SEQ ID NOS: 7 to 12).

Example 4 sensitivity of the composition of the invention

PIV-1, PIV-3 and HMPV and the spike virus samples are diluted to 1.00E+04 copies/mL, 1.00E+03 copies/mL, 4.00E+02 copies/mL, 2.00E+02 copies/mL and 1.00E+02 copies/mL in a gradient manner to serve as samples to be tested, the detection level of 95% is taken as the lowest detection limit of the kit, and analysis sensitivity test results show that the detection limits of the kit on parainfluenza virus type 1, parainfluenza virus type 3, spike virus and human metapneumovirus are respectively as follows: 200.0copies/mL, 200.0copies/mL (see FIGS. 3-6).

EXAMPLE 5 specificity of the composition of the invention

The primers and probes shown in example 1 were used to detect common infectious viruses (measles virus, mumps virus, rubella virus, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, enterovirus type 71, influenza B Victoria type, influenza A H1N1, influenza A H3N2, respiratory syncytial virus A, respiratory syncytial virus B, rhinovirus A, rhinovirus B, adenovirus type 3, adenovirus type 7) as in example 2, and the results are shown in FIG. 7, indicating no cross-reactivity with the above pathogens.

EXAMPLE 6 interference resistance and stability of the inventive composition

In order to examine the influence of possible endogenous/exogenous substances in a sample on a detection result, diluting a pathogen DNA quantitative reference with a fixed value to the minimum detection limit, dividing the pathogen DNA quantitative reference into a plurality of parts, and adding a certain concentration of endogenous interfering substances into each part: dexamethasone, cefmenoxime hydrochloride, zanamivir, ribavirin, azithromycin, histamine hydrochloride, beclomethasone, mupirocin, tobramycin, mometasone, fluticasone, budesonide, triamcinolone acetonide, heme, purified mucin and absolute ethyl alcohol with a certain concentration, and the anti-interference capability of the kit is examined by comparing the kit with a control sample. The test results are shown in Table 5. As can be seen from the table, the samples containing various interfering substances are positive through detection, and the Ct value is not obviously different from the control, so that the fact that the interfering substances possibly existing in the samples have no obvious interference on the detection result of the kit under the experimental condition of the kit can be demonstrated.

TABLE 5

Comparative example 1, remaining poorly performing primers and probes designed according to the invention

In the base complementary pairing principle, dimers are formed between the primer and (or) probe, but the probability is small and can be eliminated at the beginning of design. However, when multiple pathogens are jointly detected, a plurality of primers and probes are arranged, dimers are easy to occur between the primers and the primers, between the probes and the probes or between the primers and the probes, so that the conservation of design (which is crucial to the accuracy of detection) is ensured, and the mutual interference among different primer probes is considered, so that the primer probes need to be carefully designed and verified.

In addition, the inventors have designed that the remaining primers and probes constitute different detection systems 1, 2. The specific detection results are shown in fig. 8 or 9, and it can be seen from the graph that the detection only shows part of the amplification curve, the amplification curve has low amplification and poor repeatability, and other targets even have no amplification curve, so that the overall detection effect is poor.

Claims

1. A composition for detecting and typing respiratory viruses, comprising:

upstream and downstream primers and probes for detecting PIV-1 shown in SEQ ID NO 1-3;

an upstream primer, a downstream primer and a probe for detecting PIV-3 shown in SEQ ID NO. 4-6;

an upstream primer, a downstream primer and a probe for detecting HMPV, which are shown in SEQ ID NO. 7-9; and

and the upstream and downstream primers and probes for detecting the spike virus are shown as SEQ ID NO. 13-15.

2. The composition of claim 1, wherein the composition further comprises: and the upstream and downstream primers and probes for detecting the HMPV are shown in SEQ ID NO 10-12.

3. The composition of claim 1, wherein the fluorophores of the composition probes are different from each other and do not interfere with each other.

4. A composition according to claim 3, wherein the fluorescent reporter group of the PIV-1 probe is FAM; the fluorescence reporter group of the PIV-3 probe is HEX; the fluorescence reporter group of HPMC is CY5; the fluorescent reporter group of the wolf virus is ROX.

5. The composition according to any one of claims 1 to 4, wherein the components of the composition are present in a mixed form.

6. The composition of claim 5, wherein the composition further comprises: and detecting the upstream and downstream primers and probes of the internal standard.

7. Use of a composition according to any one of claims 1 to 6 for the preparation of a kit for detecting and typing respiratory viruses, said pathogens being PIV-1, PIV-3, HMPV and spike virus.

8. A kit for detecting and typing a respiratory virus, the kit comprising the composition of any one of claims 1-6.

9. The kit of claim 8, further comprising a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs, a DNA polymerase, a reverse transcriptase, a PCR buffer, and Mg ²⁺ At least one of (a)。

10. A method for detecting and typing a virus for non-diagnostic purposes, the method comprising the steps of:

1) Extracting or releasing nucleic acid of a sample to be tested;

2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 6 or the kit of claim 8 or 9;

3) The results were obtained and analyzed.