CN117126965A - Primer probe composition, kit and application - Google Patents
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Abstract
The application discloses a primer probe composition, a kit and application, comprising at least one of a first primer probe group for detecting novel coronavirus N gene and a second primer probe group for detecting novel coronavirus ORF1ab gene; the primer probe composition further comprises a third primer probe set for detecting influenza a virus. The application designs the first primer probe group and the second primer probe group aiming at the novel coronavirus nucleic acid specific sequence, and designs the third primer probe group aiming at the influenza A virus specific sequence, so that the co-detection of various typed novel coronaviruses and influenza A viruses can be effectively realized, the inclusion is good, and the omission detection is not easy to occur; in addition, the specificity is good, the cross reaction is not easy to occur, and false positive results are avoided; the anti-interference performance is strong, false negative results caused by inhibiting PCR amplification by interfering substances are avoided, and the detection accuracy is improved.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to a primer probe composition, a kit and application.
Background
Currently, the main known routes of spread for covd-19 include spray spread and direct contact spread, aerosol spread and indirect contact spread, with early typical clinical symptoms of fever, respiratory symptoms and muscle pain, severe cases presenting with acute respiratory distress syndrome and even acute respiratory failure.
Influenza A is mainly respiratory infectious disease caused by influenza A virus and commonly susceptible to people worldwide, is also frequently developed in winter as common seasonal influenza, is mainly transmitted by three modes of contact transmission, droplet transmission and air transmission, can cause transmission among people in a large range, and has early clinical manifestations of fever, headache, muscle pain and dyspnea, which are similar to the early onset clinical manifestations of COVID-19; moreover, laboratory examination shows that the early stage of onset blood routine of influenza patients and COVID-19 patients shows normal or reduced leucocyte, reduced lymphocyte proportion or reduced count, and the detection indexes of novel coronaviruses and influenza viruses are indicated to show similar changes.
In view of this, the related art uses real-time quantitative nucleic acid amplification (real time qPCR) detection to detect common nucleic acids of the novel coronavirus and influenza a virus, which has the main advantages of fast detection speed and high detection sensitivity to detect the novel coronavirus infection and influenza a virus conveniently and efficiently, but in detecting clinical samples, the novel coronavirus and influenza a virus have multiple types, such as original strain, alpha strain, beta strain, delta strain, omicton strain and the like of the common novel coronavirus, H1N1 (seasonal), H1N1 (pdm 09), H3N2, H5N1, H7N9 and the like of the common influenza a virus, and the coverage of the primers/probes is insufficient, so that the inclusion is poor, the detection omission is easy to occur, and in addition, when two or three viruses exist in the samples simultaneously, the mutual influence between the respective primers may cause performance degradation, so that the virus amplification is interfered with each other.
Disclosure of Invention
Aiming at the problems in the prior art, the application provides a primer probe composition, a kit and application, the primers can not interfere with each other, the novel coronavirus with multiple types can be effectively detected, the inclusion is good, the detection omission is not easy to occur, and the detection accuracy is improved. The technical scheme is as follows:
the application provides a primer probe composition, which comprises at least one of a first primer probe group for detecting a novel coronavirus N gene and a second primer probe group for detecting a novel coronavirus ORF1ab gene;
the first primer probe group comprises an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2 and a probe shown as SEQ ID NO. 3;
the second primer probe group comprises an upstream primer shown as SEQ ID NO.4, a downstream primer shown as SEQ ID NO.5 and a probe shown as SEQ ID NO. 6;
the primer probe composition also comprises a third primer probe group for detecting influenza A virus, wherein the third primer probe group comprises an upstream primer shown as SEQ ID NO.7, a downstream primer shown as SEQ ID NO.8 and a probe shown as SEQ ID NO. 9.
Further, the primer probe composition also comprises a fourth primer probe group for detecting the influenza B virus,
the fourth primer probe group comprises an upstream primer shown as SEQ ID NO.10, a downstream primer shown as SEQ ID NO.11 and a probe shown as SEQ ID NO. 12.
Further, the probes of the first primer probe group, the probes of the second primer probe group, the probes of the third primer probe group and the probes of the fourth primer probe group adopted by the primer probe composition are respectively marked by adopting modification groups showing different fluorescent colors.
Further, the modifying group includes a fluorescent group and a quenching group;
the fluorescent group comprises at least one of a Cy5 fluorescent group, a VIC fluorescent group, a FAM fluorescent group and a ROX fluorescent group;
the quenching group comprises at least one of a TAMRA quenching group, an MGB quenching group, a BHQ1 quenching group and a BHQ2 quenching group.
The application also provides a kit comprising qPCR premix, reverse transcriptase and the primer probe composition described in any one of the above.
Further, the kit satisfies at least one of the following characteristics:
at least one of the upstream primer, the downstream primer and the probe of the first primer probe group has the concentration of 70-150 mug/mL;
At least one of the upstream primer, the downstream primer and the probe of the second primer probe set has a concentration of 70-150 mug/mL;
at least one of the upstream primer, the downstream primer and the probe of the third primer probe group has the concentration of 70-150 mug/mL;
at least one of the upstream primer, the downstream primer and the probe of the fourth primer probe set has a concentration of 70-150 mug/mL.
The application also provides application of the primer probe composition or the kit in detection of novel coronaviruses and influenza A viruses.
The application also provides application of the primer probe composition or the kit in detecting at least one of influenza A virus and influenza B virus and novel coronavirus.
Further, the primer probe composition or the kit is applied to a detection method of novel coronaviruses and influenza A viruses, the adopted PCR amplification reaction comprises a reverse transcription stage, a pre-denaturation stage, a denaturation stage and an annealing stage, and the preset conditions of the PCR amplification reaction comprise at least one of the following conditions:
the preset reverse transcription temperature of the reverse transcription stage is 40-50 ℃;
The preset pre-denaturation temperature of the pre-denaturation stage is 90-97 ℃;
the preset denaturation temperature of the denaturation stage is 90-97 ℃;
the preset annealing temperature of the annealing stage is 45-60 ℃;
the preset cycle number of the PCR amplification reaction is 30-50.
Further, the kit comprises reverse transcriptase, the kit is applied to a detection method of novel coronaviruses and influenza A viruses, and the dosage of the reverse transcriptase adopted in the kit is 15U-40U in a single detection process.
Further, the use of the primer probe composition or the kit satisfies at least one of the following characteristics:
the lowest detection limit of the primer probe composition or the kit against the novel coronavirus is 15-150 copies/mL;
the lowest detection limit of the primer probe composition or the kit against influenza A virus is 30-150 copies/mL;
the lowest detection limit of the primer probe composition or the kit against the influenza B virus is 150-750 copies/mL.
Further, the lowest detection limit of the primer probe composition or the kit for influenza A virus and/or influenza B virus is 10 in dilution of stock solution after the national reference is reconstituted 5 ～10 6 Concentration after doubling.
Further, the coefficient of variation of the cycle number threshold value of the primer probe composition or the kit applied to the detection method of the novel coronavirus and the influenza A virus is less than 5%.
Further, the primer probe composition or the kit has a significance probability of greater than 0.05 when applied to a detection method of novel coronaviruses and influenza a viruses.
The implementation of the application has the following beneficial effects:
1. the application designs the first primer probe group and the second primer probe group aiming at the novel coronavirus nucleic acid specific sequence, has high coverage for the novel coronavirus, can effectively detect the novel coronaviruses with different types, improves the inclusion of the novel coronavirus detection, and simultaneously designs the third primer probe group aiming at the influenza A virus specific sequence, has high coverage for the influenza A virus, can detect the influenza A viruses with different types and is not easy to leak detection; the first primer probe set, the second primer probe set and the third primer probe set have no mutual interference, and the respective PCR amplification is not affected; in addition, the first primer probe set and the second primer probe set have good specificity for the novel coronavirus, are not easy to have cross reaction with colonization bacteria or other pathogens possibly existing in the sample, so that false positive detection is avoided; in addition, the first primer probe group and the second primer probe group are high in anti-interference performance, false negative caused by the fact that interference substances in a sample inhibit PCR (polymerase chain reaction) amplification is avoided, and detection accuracy is greatly improved.
2. The primer probe composition or the kit provided by the application has the advantages that the primer probe composition or the kit is high in detection effectiveness, good in specificity, good in inclusion and good in accuracy for each of novel coronavirus, influenza A virus and influenza B virus, and can be used for identifying the three viruses in one detection, so that repeated detection for multiple times is avoided, a patient is subjected to targeted treatment, the serious illness is reduced, the death rate is reduced, the exposure risk of communities and medical staff is reduced, the cross infection of pneumonitis patients is reduced, and unnecessary panic and resource waste are avoided.
3. The first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set have no mutual interference, do not influence PCR amplification, and can greatly improve the detection accuracy and the detection reliability of the novel coronavirus, the influenza A virus and the influenza B virus during co-detection.
4. The primer probe composition or the kit has the advantages of small dosage and high detection sensitivity, and the detection sensitivity for the novel coronaviruses, the influenza A viruses and the influenza B viruses is far higher than the requirements of corresponding national references.
Drawings
In order to more clearly illustrate the technical solution of the present application, the following description will briefly explain the drawings used in the embodiments, in which like elements are denoted by like reference numerals. It is evident that the drawings in the following description are only some embodiments of the present application and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a graph showing the results of detection of novel coronaviruses using primer probe compositions of the present application;
FIG. 2 shows the detection results of influenza A virus using a primer probe composition according to the present application;
FIG. 3 shows the detection result of the application for detecting influenza B virus using a primer probe composition;
FIG. 4 shows the results of the co-detection of novel coronaviruses, influenza A viruses and influenza B viruses using the primer probe composition of the present application.
Detailed Description
The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and thus should not be construed as limiting the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
It is noted that in the description of the present application, for the terms defined below, these definitions shall be applied unless a different definition is given in the claims or elsewhere in this specification. All numerical values, whether or not explicitly indicated, are defined herein as modified by the term "about". The term "about" generally refers to a range of values that one of ordinary skill in the art would consider equivalent to the stated value to produce substantially the same properties, functions, results, etc. A range of values indicated by a low value and a high value is defined to include all values included within the range of values and all subranges included within the range of values.
It should be noted that the terms "first," "second," and the like in the description and the claims and drawings of the present application are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the objects so used are interchangeable under appropriate circumstances such that the embodiments of the application are capable of operation in other sequences than described of illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or article that comprises a list of steps or elements is not necessarily limited to those steps or elements that are expressly listed or inherent to such process, method, or article.
Influenza B is mainly respiratory infectious disease which is caused by influenza B virus and is commonly and easily felt by people in the world, is similar to influenza A, is also frequently developed in winter as common seasonal influenza, is mainly transmitted by means of three modes of contact transmission, droplet transmission and air transmission, can cause transmission among people in a large range, and early clinical manifestations of influenza A and influenza B patients are mainly fever, headache, muscle pain and dyspnea, and is similar to early onset clinical manifestations of COVID-19; moreover, laboratory examination shows that the early stage of onset blood routine of patients with influenza A and influenza B and patients with COVID-19 shows normal or reduced leucocytes, reduced lymphocyte proportion or reduced count, and the detection indexes of the novel coronavirus, influenza A virus and influenza B virus are indicated to show similar changes.
The clinical manifestations, transmission routes and season of influenza A, influenza B and novel coronavirus infection are similar, and in patients in non-severe epidemic areas, the confirmed diagnosis of SARS-CoV-2 infection can still be combined with influenza virus infection, so that the differential diagnosis in one detection is very important; simultaneous detection of novel coronavirus infection and influenza a/b can be conveniently achieved using multiplex fluorescence PCR, but when clinical samples are detected using multiplex fluorescence PCR, the following problems are often encountered:
1) The mutual interference between the primers: the targets are amplified singly, but the targets are combined to have influence on each other, so that the performance is reduced, and when two or three viruses exist in a sample at the same time, the amplification of the targets can be interfered with each other;
2) The novel coronavirus and the influenza virus are all of various types, and the novel coronavirus and the influenza virus are all of various types, such as original strain, alpha strain, beta strain, delta strain, omicron strain and the like; common influenza a viruses are H1N1 (seasonal), H1N1 (pdm 09), H3N2, H5N1, H7N9, etc.; common influenza B viruses include Victoria, yamagata, etc., and the coverage of the primer/probe is insufficient, so that the inclusion is poor and the omission is easy to occur;
3) The respiratory tract contains a large number of colonization bacteria and other pathogens, the specificity of the primer/probe is poor, and cross reaction is easy to occur, so that a false positive result is obtained;
4) Detection of novel coronavirus infection and influenza, typically using a pharyngeal swab or a nasopharyngeal swab, interfering substances in the sample may inhibit the PCR reaction, leading to false negative results.
In order to solve at least one of the problems faced in the multiplex fluorescence PCR detection process, the application provides a primer probe composition and a kit, wherein the primer probe composition comprises at least one of a first primer probe set for detecting novel coronavirus N genes, a second primer probe set for detecting novel coronavirus ORF1ab genes, a third primer probe set for detecting influenza A viruses and a fourth primer probe set for detecting influenza B viruses, and primer sequences and probe sequences in the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set are shown in table 1.
TABLE 1 sequence of primer probe compositions
In a preferred embodiment, the first primer probe set and the second primer probe set in the primer probe composition are used for detecting the novel coronavirus so as to carry out co-detection on the novel coronavirus and improve the inclusion of detection; for an unknown sample, the first primer probe set and the second primer probe set are adopted for detection, wherein the detection result of any one set is positive, the sample is judged to be positive for the novel coronavirus, namely, the sample can be judged to be negative for the novel coronavirus only if the detection results of the first primer probe set and the second primer probe set are negative.
The detection results of the first primer probe set and the second primer probe set specifically comprise the following four types:
firstly, adopting a first primer probe set to detect a positive detection result of a sample, and adopting a second primer probe set to detect a negative detection result, and judging that the sample is positive to the novel coronavirus;
secondly, the detection result of detecting the sample by adopting the second primer probe set is positive, and the detection result of detecting the sample by adopting the first primer probe set is negative, so that the sample is judged to be positive for the novel coronavirus;
thirdly, adopting the first primer probe group and the second primer probe group to detect the sample respectively, and judging that the sample is positive to the novel coronavirus if the detection results are positive;
Fourth, the first primer probe group and the second primer probe group are adopted to detect the sample respectively, and the detection results are negative, so that the sample is judged to be negative to the novel coronavirus.
The primer probe composition comprising the first primer probe set and the second primer probe set has high coverage to the novel coronavirus, can effectively detect the novel coronavirus with different types, has good inclusion, is not easy to leak detection, and has high detection accuracy.
Specifically, the primer probe composition further comprises a third primer probe group for detecting the influenza A virus, the novel coronavirus and the influenza A virus are detected simultaneously through the first primer probe group, the second primer probe group and the third primer probe group, wherein the first primer probe group and the second primer probe group have good specificity and inclusion for the novel coronavirus, the third primer probe group has good specificity and inclusion for the influenza A virus, the three primer probe groups have no mutual interference, and when a plurality of viruses exist in a sample simultaneously, the respective amplifications cannot mutually interfere, and the detection accuracy is high.
In other alternative embodiments, the primer probe composition comprises a first primer probe group and a second primer probe group, the primer probe composition further comprises a fourth primer probe group for detecting the influenza b virus, the simultaneous detection of the novel coronavirus and the influenza b virus is realized through the first primer probe group, the second primer probe group and the fourth primer probe group, wherein the first primer probe group and the second primer probe group have good specificity and inclusion for the novel coronavirus, the fourth primer probe group has good specificity and inclusion for the influenza b virus, the three primer probe groups have no mutual interference, and when multiple viruses exist in a sample at the same time, the respective amplifications can not mutually interfere, and the detection accuracy is high.
In some preferred embodiments, the primer probe composition comprises a first primer probe group, a second primer probe group, a third primer probe group and a fourth primer probe group, so that the co-detection of novel coronaviruses, influenza A viruses and influenza B viruses is realized, the positive or negative of the novel coronaviruses and the influenza A/B viruses can be accurately detected through one-time detection, the types of viruses in a sample are identified, the specificity is good, the inclusion is good, the anti-interference capability is strong, the detection efficiency is high, the reliability is high, and the accuracy is high; in addition, the specificity of the four primer probe sets is very excellent, and cross reaction with the colonization bacteria and other pathogens in the sample is not easy to occur, so that false positive results are avoided; in addition, the four primer probe groups are not easily affected by interference substances in the sample, so that false negative results caused by the inhibition of PCR amplification reaction by the interference substances are avoided, and the detection accuracy is greatly improved; the primer probe composition is used for detection, can be used for differential diagnosis in one detection process, enables patients to be treated in a targeted manner, is favorable for reducing severe symptoms and mortality, can reduce exposure risks of communities and medical staff, reduces cross infection of patients suffering from pneumonia, and avoids unnecessary panic and resource waste.
Specifically, the probes of the first primer probe group, the probes of the second primer probe group, the probes of the third primer probe group and the probes of the fourth primer probe group adopted by the primer probe composition are respectively marked by adopting modification groups showing different fluorescent colors so as to effectively distinguish detection results corresponding to different primer probe groups and avoid confusion and false detection; wherein the modifying group comprises a fluorescent group and a quenching group, the fluorescent group comprises at least one of a Cy5 fluorescent group, a VIC fluorescent group, a FAM fluorescent group and a ROX fluorescent group, and the quenching group comprises at least one of a TAMRA quenching group, an MGB quenching group, a BHQ1 quenching group and a BHQ2 quenching group; it should be noted that the modifying groups are not limited to the above-listed fluorescent groups and quenching groups, and may include other fluorescent groups and quenching groups, so long as the modifying groups capable of effectively distinguishing different probes are within the scope of the present disclosure.
The application also provides a kit, which comprises qPCR premix (qPCR mix), reverse transcriptase and the primer probe composition, wherein the qPCR mix is a ready-to-use mixture and comprises all components except a primer and a template, and is used for amplifying and detecting nucleic acid in the qPCR process; in the application, the qPCR mix comprises all components except a primer, a template and reverse transcriptase, and is used for fluorescent PCR amplification of novel coronaviruses, influenza A viruses and/or influenza B viruses; reverse transcriptase is used to catalyze the polymerization of deoxyribonucleoside triphosphates to DNA using viral RNA as a template.
Specifically, in some alternative embodiments, the concentration of at least one of the upstream primer of the first primer probe set, the downstream primer and the probe in the kit is 70-150 μg/mL; it will be appreciated that the concentration of the upstream primer, the downstream primer and the probe in the first primer probe set may be any point value from 70. Mu.g/mL to 150. Mu.g/mL, and may be, for example, 70. Mu.g/mL, 80. Mu.g/mL, 100. Mu.g/mL, 120. Mu.g/mL, 130. Mu.g/mL, 150. Mu.g/mL, etc., and the concentrations of the upstream primer, the downstream primer and the probe may be selected to be the same or different; when in use, the upstream primer, the downstream primer and the probe with preset amounts can be removed for detection, thereby being convenient and quick.
In some alternative embodiments, the concentration of at least one of the upstream primer, the downstream primer, and the probe in the second primer-probe set in the kit is 70-150. Mu.g/mL, i.e., the concentration of the upstream primer, the downstream primer, and the probe in the second primer-probe set may be any point value in the range of 70. Mu.g/mL-150. Mu.g/mL, not enumerated herein.
In some alternative embodiments, the concentration of at least one of the upstream primer, the downstream primer, and the probe of the third primer probe set in the kit is 70 to 150 μg/mL; in some alternative embodiments, the concentration of at least one of the upstream primer, the downstream primer, and the probe of the fourth primer probe set in the kit is 70 to 150 μg/mL.
Specifically, the kit stores primer probe compositions with preset volumes respectively, wherein the preset volumes are larger than or equal to the preset dosage of the primer probe compositions in a single detection process; in an alternative embodiment, the preset volume is larger than the preset amount of the primer probe composition in the single detection process, and can be used in the multiple detection processes, wherein the preset amount of the primer probe composition corresponding to the single detection is moved in the single detection process for detection; preferably, the preset volume is equal to the preset dosage of the primer probe composition in the single detection process, namely each kit is used for single detection, and the external environment pollution kit in the multiple opening and closing processes is avoided.
The application also provides application of the primer probe set or the kit in detecting novel coronaviruses and influenza A viruses, which has the advantages of strong specificity, good inclusion, difficult interference and greatly improved detection efficiency and detection accuracy.
The application also provides application of the primer probe composition or the kit in detecting at least one of influenza A virus and influenza B virus and novel coronavirus, which can be independently used for detecting the novel coronavirus, the influenza A virus and the influenza B virus; further, the method can be used for co-detection of two viruses in the three viruses, for example, the novel coronavirus and the influenza A virus, and can also be used for co-detection of the novel coronavirus, the influenza A virus and the influenza B virus, and has the advantages of strong specificity, good inclusion, difficult interference and great improvement of detection efficiency and detection accuracy.
In an alternative embodiment, the primer probe composition or kit is applied to a detection method of a novel coronavirus and an influenza a virus, or to a detection method of co-detecting at least one of an influenza a virus and an influenza b virus with a novel coronavirus, and in a single detection process, the preset amount of each upstream primer is 0.1 μl to 2 μl, the preset amount of each downstream primer is 0.1 μl to 2 μl, the preset amount of each probe is 0.1 μl to 2 μl, that is, the preset amount of each upstream primer, downstream primer and probe is optionally selected to be any point value of 0.1 μl to 2 μl, and may be, for example, 0.1 μl,0.2 μl,0.25 μl,0.5 μl,0.75 μl,1.5 μl,2 μl, etc.
Specifically, in an alternative embodiment, the kit is applied to a novel coronavirus and an influenza a virus, or applied to a detection method for co-detecting at least one of an influenza a virus and an influenza b virus with the novel coronavirus, and the amount of reverse transcriptase adopted in the kit is 15U to 40U in a single detection process, and may be 15U,20U,30U,35U,40U, or the like, for example.
Specifically, in an alternative embodiment, the kit is applied to a detection method of novel coronaviruses and influenza A viruses, or a detection method of co-detecting at least one of influenza A viruses and influenza B viruses and the novel coronaviruses, and in a single detection process, the using amount of qPCR mix adopted in the kit is 5-20 mu L; it will be appreciated that the qPCR mix may be used in an amount of any point from 5. Mu.L to 20. Mu.L, and may be, for example, 5. Mu.L, 8. Mu.L, 10. Mu.L, 15. Mu.L, 20. Mu.L, etc.
Optionally, the kit further comprises purified water, wherein the purified water is pure matter comprising and only comprising hydrogen oxide, so that the convenience of using the kit is improved.
Specifically, the primer probe composition or the kit is applied to the novel coronavirus and the influenza A virus or applied to a detection method for co-detecting at least one of the influenza A virus and the influenza B virus and the novel coronavirus, and a PCR amplification reaction is adopted, wherein the PCR amplification reaction comprises a reverse transcription stage, a pre-denaturation stage, a denaturation stage and an annealing stage, and then the preset conditions of the PCR amplification reaction comprise at least one of the following conditions:
the reverse transcription temperature in the reverse transcription stage is 40-50 ℃;
the pre-denaturation temperature in the pre-denaturation stage is 90-97 ℃;
the denaturation temperature in the denaturation stage is 90-97 ℃;
the annealing temperature in the annealing stage is 45-60 ℃;
the number of cycles of PCR amplification is 30 to 50.
It will be appreciated that the temperature in the above-mentioned preset conditions may be selected to be a point value, for example, a reverse transcription temperature of 42 ℃; alternatively, a range of values, such as a pre-denaturation temperature of 94℃to 96℃may be used to maintain a dynamic stability of the temperature and a stable PCR amplification reaction.
In the present application, the minimum limit of detection (Limit of Detection, LOD) refers to the minimum content or minimum concentration of viral target sequences that can be detected under specific experimental conditions; the primer probe composition or the kit has the lowest detection limit for the novel coronaviruses, the influenza A viruses and the influenza B viruses far lower than the requirements of corresponding national references, wherein the lowest detection limit for the novel coronaviruses is 15-150 copies/mL; it will be appreciated that the minimum detection limit for the novel coronavirus may be any point value in the range of 15 to 150copies/mL, and may be, for example, 15copies/mL,30copies/mL,50copies/mL,70copies/mL,100copies/mL,120copies/mL,150copies/mL, etc.; in some embodiments, the minimum detection limit for the novel coronavirus is 30-150 copies/mL, and the detection sensitivity is high.
The lowest detection limit for influenza A virus and influenza B virus is 10 dilution of stock solution after the national reference is redissolved 5 ～10 6 The concentration after doubling, wherein the minimum detection limit for influenza A virus is 30-150 copies/mL; it will be appreciated that the minimum detection limit for influenza A virus is any point value in the range of 30 to 150copies/mL, and may be, for example, 30copies/mL,50copies/mL,70copies/mL,80copies/mL,100copies/mL,120copies/mL,150copies/mL, etc., with high detection sensitivity, and is notAnd the omission is easy to occur.
The minimum detection limit for the influenza B virus is 150-750 copies/mL; it can be understood that the lowest detection limit for the influenza B virus is any point value in 150-750 copies/mL, and can be, for example, 150copies/mL,200copies/mL,260copies/mL,350copies/mL,500copies/mL,600copies/mL,700copies/mL,750copies/mL, and the like, so that the detection sensitivity is high, and the detection result is accurate and reliable.
The technical scheme of the application is described in detail below with reference to specific embodiments.
Example 1 inclusion verification
The primer probe composition with the nucleotide sequence shown in the table 1 is adopted in the embodiment to verify the inclusion of the primer probe composition comprising four primer probe sets against novel coronaviruses, influenza a viruses and influenza b viruses; the 5 'end of the first primer probe group probe is marked by a Cy5 fluorescent group, and the 3' end of the first primer probe group probe is marked by a BHQ2 quenching group; marking the 5 'end of the second primer probe set probe by a VIC fluorescent group, and marking the 3' end by a BHQ1 quenching group; marking the 5 'end of the third primer probe set probe by FAM fluorescent groups and marking the 3' end by BHQ1 quenching groups; the 5 'end of the fourth primer probe set probe was labeled with a ROX fluorophore and the 3' end thereof was labeled with a BHQ2 quencher.
The kit is used in the detection method of novel coronaviruses, influenza A viruses and influenza B viruses, and the reaction system adopted in the kit in the single detection process is shown in the following table 2.
TABLE 2 reaction System for Single-pass detection procedure of novel coronavirus Single target
| Name of the name | Preset amount of |
| N-F(100μg/mL) | 0.75μL |
| N-R(100μg/mL) | 0.75μL |
| N-P(100μg/mL) | 0.25μL |
| ORF1ab-F(100μg/mL) | 0.2μL |
| ORF1ab-R(100μg/mL) | 0.2μL |
| ORF1ab-P(100μg/mL) | 0.2μL |
| FluA-F(100μg/mL) | 0.25μL |
| FluA-R(100μg/mL) | 0.25μL |
| FluA-P(100μg/mL) | 0.25μL |
| FluB-F(100μg/mL) | 0.25μL |
| FluB-R(100μg/mL) | 0.25μL |
| FluB-P(100μg/mL) | 0.25μL |
| qPCR mix(2×) | 10μL |
| Reverse transcriptase | 20U |
| Purified water | Make up 20 mu L |
The primer/probe can be synthesized by any company with related synthesis business, such as Thermo, takara, AG, a worker and the like; the 2 XqPCR mix can be conventional commercially available qPCR mix such as Thermo, takara, meridian, northenan, etc., and can also be prepared by the experimenter; the reverse transcriptase may be a conventional commercially available reverse transcriptase such as Thermo, takara, AG, novzan, etc.
In this reaction system, the preset conditions for the PCR amplification reaction are shown in Table 3 below.
TABLE 3 preset conditions for PCR amplification reactions
In fluorescence PCR amplification, the number of target sequences in a reaction system is gradually increased along with the increase of the number of reaction cycles after multiple amplifications, when the number of target sequences reaches a certain degree, a fluorescence signal exceeds a set threshold line, the number of circulation cycles can be recorded at the moment and is called a cycle number threshold (Cycle Threshold Value, ct), the smaller the Ct value is, the richer the target sequences in the reaction system are, the faster the amplification speed is, and the relative difference of the number of target sequences in different samples can be deduced by calculating the Ct value difference between samples; the coefficient of variation CV is also called standard deviation, which is a statistic for measuring the variation degree of each detection value in a sample and reflects the discrete degree on a unit mean, and the smaller the coefficient of variation is, the higher the precision is.
Samples were diluted using 20 samples of novel coronavirus pharyngeal swabs from different regions and 16 novel coronavirus strains (containing all common lineages), repeatedly tested 10 times under the reaction system shown in table 2 and the preset conditions shown in table 3, and the CV of Ct value was calculated to verify its inclusion and precision.
TABLE 4 results of inclusion verification of the primer probe composition against novel coronavirus samples
TABLE 5 results of inclusion verification of the primer probe composition against novel coronavirus strains
As shown in the verification results in table 4 and table 5, after 20 novel coronavirus pharyngeal swab samples and 16 novel coronavirus strains are diluted, the detection is repeated 10 times, the results show that the samples are positive, and the computed CV of the Ct value is less than 5%, which indicates that the primer probe composition has good inclusion for the novel coronavirus.
The samples were diluted using 21 samples/strains (containing all common subtypes) of influenza a virus from different regions, repeatedly tested 10 times under the reaction system shown in table 2 and the preset conditions shown in table 3, and the CV of Ct value was calculated to verify its inclusion and precision.
TABLE 6 results of inclusion verification of the primer probe composition against influenza A virus
The verification results in Table 6 show that CV of Ct value obtained by repeated detection for 10 times after dilution of 21 influenza A virus samples/strains is less than 5%, which indicates that the primer probe composition has good inclusion for influenza A virus.
The inclusion was verified by repeating 10 times the test using 20 samples/strains of influenza b virus from different regions (10 samples each of two common lineages) in the reaction system shown in table 2 and the preset conditions shown in table 3 after diluting the samples.
TABLE 7 results of inclusion verification of the primer probe composition against influenza B virus
| Sample/strain numbering | Pedigree of pedigree | Origin place | Precision of |
| CL-NT016 | Victoria | C province | 2.97％ |
| CL-NT017 | Victoria | C province | 1.19％ |
| CL-NT018 | Victoria | C province | 0.68％ |
| CL-NT019 | Victoria | C province | 1.28％ |
| CL-LZ016 | Victoria | D province | 0.95％ |
| CL-LZ017 | Victoria | D province | 0.56％ |
| CL-LZ018 | Victoria | D province | 2.97％ |
| CL-LZ019 | Victoria | D province | 1.15％ |
| CL-NC016 | Victoria | B province | 0.74％ |
| CL-NC017 | Victoria | B province | 1.16％ |
| CL-NT011 | Yamagata | C province | 0.94％ |
| CL-NT012 | Yamagata | C province | 0.87％ |
| CL-NT013 | Yamagata | C province | 0.53％ |
| CL-NT014 | Yamagata | C province | 0.82％ |
| CL-LZ011 | Yamagata | D province | 0.60％ |
| CL-LZ012 | Yamagata | D province | 2.84％ |
| CL-LZ013 | Yamagata | D province | 0.77％ |
| CL-NC013 | Yamagata | B province | 2.99％ |
| CL-NC014 | Yamagata | B province | 1.21％ |
| CL-NC015 | Yamagata | B province | 0.73％ |
The verification results in Table 7 show that 20 influenza B virus samples/strains are diluted to 5 XLOD concentration, the repeated detection results are positive for 10 times, and CV of Ct values obtained by calculation are smaller than 5%, which indicates that the primer probe composition has good inclusion for influenza B virus.
Example 2 verification of interference immunity between Single and Multi-targets
Preparing four reaction systems by using primer probe compositions shown in table 1, respectively performing single-target detection and multi-target detection according to preset conditions shown in table 3, extracting nucleic acid from a mixed sample by a magnetic bead method, wherein the mixed sample comprises 5.0X10 3 Influenza A virus H1N1, 5.0X10/mL 3 Influenza B virus Yamagata and 5.0X10 of copies/mL 3 The novel coronavirus 2019-nCoV of cobies/mL; the four reaction systems comprise a first reaction system for carrying out single-target detection on the novel coronavirus by adopting a first primer probe composition and a second primer probe composition, a second reaction system for carrying out single-target detection on the influenza A virus by adopting a third primer probe composition, and a first reaction system for carrying out single-target detection on the influenza B virus by adopting a fourth primer probe compositionAnd a third reaction system, which adopts a fourth reaction system for multi-target detection by the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set.
Wherein, the detection result of the first reaction system is shown in fig. 1, the detection result of the second reaction system is shown in fig. 2, the detection result of the third reaction system is shown in fig. 3, and the detection result of the fourth reaction system is shown in fig. 4; therefore, when the primer probe sets are used singly and in combination, the PCR amplification curve of each detection target is not obviously changed, which indicates that in the primer probe composition, four groups of primers cannot interfere with each other in the PCR amplification process, and the performance is excellent.
Further, ct values for single-target and multi-target assays are recorded as shown in table 8 below.
Table 8 Ct values for each primer probe set used alone and in combination
As can be seen by comparison, when the primer probe sets are used singly and in combination, the target CT values of the primer probe sets are almost not different, namely, the detection quantity of various virus target sequences is almost not different in different reaction systems, which indicates that the primers in the first primer probe set, the primers in the second primer probe set, the primers in the third primer probe set and the primers in the fourth primer probe set can not interfere with each other in the PCR amplification process.
Example 3 sensitivity and minimum detection limit verification
The reaction system shown in Table 2 was formulated using the four primer probe sets shown in Table 1, and the minimum detection limit was verified using national references.
First, the sensitivity to the novel coronavirus is verified; according to the requirements of the national reference for nucleic acid detection reagent of 2019-nCoV, the sensitivity reference S is diluted by 1:3 times (2 parts of water and 1 part of sample) with deionized water without RNA/DNase, and the sensitivity reference S is respectively marked as S1-S10 by 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, 1:6561, 1:19683, 1:59049 and 1:177147, and the nucleic acid extraction kit (magnetic bead method) of Chuangla biological technology Co., suzhou is used for carrying out nucleic acid extraction and detection; according to the requirements of national reference, the detection results of S1-S3 are positive, and S4-S10 are not required.
TABLE 9 detection results of novel coronavirus national reference using the primer probe composition of the present application
The detection results of the S1-S10 reference products are shown in Table 9, and the first primer probe group and the second primer probe group in the primer probe composition can effectively detect novel coronaviruses in the S1-S7 reference products, and the sensitivity is far higher than the requirements of national reference products.
Secondly, the sensitivity against influenza a virus and influenza b virus is verified; according to the requirements of the specification of the national reference for second generation influenza A/B virus nucleic acid detection reagent, adding 0.5mL of deionized water without RNase into the reference S1-S5 with the lowest detection limit, re-dissolving, and respectively carrying out 1:10 with the deionized water without RNase 2 、1:10 3 、1:10 4 、1:10 5 1:10 6 And (5) diluting by times.
TABLE 10 detection results of national references for influenza A/B virus using the primer probe compositions of the present application
The detection results are shown in table 10, and it can be seen that the third primer probe set and the fourth primer probe set in the primer probe composition can effectively detect influenza a virus and influenza b virus, and the minimum detection limit for influenza a virus and the minimum detection limit for influenza b virus are both based on meeting the national reference requirements, and can be diluted by 10-100 times, and the sensitivity is far higher than the national reference requirements.
Further, verifying the inclusion of the primer probe composition for the virus sample at the lowest detection limit concentration; wherein, 20 samples of novel coronavirus pharyngeal swab from different regions and 16 novel coronavirus strains (containing all common lineages) were used, the samples were diluted to a 1×lod concentration, the detection was repeated 20 times, and the lowest detection limit was verified.
TABLE 11 results of inclusion verification of the primer probe composition against novel coronavirus samples at minimum detection limit concentration
TABLE 12 results of inclusion verification of the primer probe composition against novel coronavirus strains at minimum detection limit concentration
As shown in the verification results in tables 11 and 12, 20 novel coronavirus throat swab samples and 16 novel coronavirus strains are diluted to the concentration of 1 XLOD, and the detection is repeated for 20 times, and the results are positive, so that the primer probe composition has good inclusion even for the novel coronavirus at the lowest detection limit concentration.
The minimum detection limit was verified using 21 influenza a virus samples/strains (containing all common subtypes) from different regions, diluting the samples to a 1 xlod concentration, repeating the detection 20 times.
TABLE 13 results of inclusion verification of the primer probe composition against influenza A at minimum detection limit concentration
| Sample/strain numbering | Subtype type | Origin place | Detection limit |
| CL-NC002 | H1N1(pdm09) | B province | 20/20 |
| CL-NC003 | H1N1(pdm09) | B province | 20/20 |
| CL-LZ001 | H1N1(pdm09) | D province | 20/20 |
| CL-LZ002 | H1N1(pdm09) | D province | 20/20 |
| CL-LZ003 | H1N1(pdm09) | E province | 20/20 |
| DZ-H1N1-08 | H1N1 (seasonal) | E province | 20/20 |
| DZ-H1N1-09 | H1N1 (seasonal) | E province | 20/20 |
| DZ-H1N1-10 | H1N1 (seasonal) | A province | 20/20 |
| CL-NT006 | H3N2 | C province | 20/20 |
| CL-NT007 | H3N2 | C province | 20/20 |
| CL-NT008 | H3N2 | C province | 20/20 |
| CL-NC006 | H3N2 | D province | 20/20 |
| CL-NC007 | H3N2 | D province | 20/20 |
| CL-LZ006 | H3N2 | B province | 20/20 |
| CL-LZ007 | H3N2 | B province | 20/20 |
| DZ-H5N1-08 | H5N1 | E province | 20/20 |
| DZ-H5N1-09 | H5N1 | C province | 20/20 |
| DZ-H5N1-10 | H5N1 | F province | 20/20 |
| DZ-H7N9-08 | H7N9 | C province | 20/20 |
| DZ-H7N9-09 | H7N9 | E province | 20/20 |
| DZ-H7N9-10 | H7N9 | B province | 20/20 |
The verification results in table 13 show that 21 influenza a virus samples/strains are diluted to a concentration of 1×lod, and the repeated detection results for 20 times are positive, which indicates that the primer probe composition has good inclusion even for influenza a virus at the lowest detection limit concentration.
Samples were diluted to 1 x LOD concentration using 20 samples/strains of influenza b virus from different regions (10 each of the two common lineages) and repeated 20 times to verify their minimum detection limit.
TABLE 14 results of inclusion verification of the primer probe composition against influenza B virus at minimum detection limit concentration
| Sample/strain numbering | Pedigree of pedigree | Origin place | Detection limit |
| CL-NT016 | Victoria | C province | 20/20 |
| CL-NT017 | Victoria | C province | 20/20 |
| CL-NT018 | Victoria | C province | 20/20 |
| CL-NT019 | Victoria | C province | 20/20 |
| CL-LZ016 | Victoria | D province | 20/20 |
| CL-LZ017 | Victoria | D province | 20/20 |
| CL-LZ018 | Victoria | D province | 20/20 |
| CL-LZ019 | Victoria | D province | 20/20 |
| CL-NC016 | Victoria | B province | 20/20 |
| CL-NC017 | Victoria | B province | 20/20 |
| CL-NT011 | Yamagata | C province | 20/20 |
| CL-NT012 | Yamagata | C province | 20/20 |
| CL-NT013 | Yamagata | C province | 20/20 |
| CL-NT014 | Yamagata | C province | 20/20 |
| CL-LZ011 | Yamagata | D province | 20/20 |
| CL-LZ012 | Yamagata | D province | 20/20 |
| CL-LZ013 | Yamagata | D province | 20/20 |
| CL-NC013 | Yamagata | B province | 20/20 |
| CL-NC014 | Yamagata | B province | 20/20 |
| CL-NC015 | Yamagata | B province | 20/20 |
The verification results in table 14 show that 20 influenza b virus samples/strains are diluted to a 1 xlod concentration, and the repeated detection results of 20 times are positive, which indicates that the primer probe composition has good inclusion even for influenza b virus at the lowest detection limit concentration; therefore, even if the virus target sequence in the sample is at the lowest detection limit, the primer probe composition can detect viruses with different types, and has extremely high sensitivity and excellent inclusion.
Example 4 specificity verification (Cross-reaction)
The four primer probe sets in example 1 were used to formulate a reaction system as shown in table 2 and set up preset conditions as shown in table 3 to verify the cross-reactivity of the primer probe composition with common pathogens in the respiratory tract during PCR amplification.
TABLE 15 results of specificity verification of the primer probe composition
As shown in table 15, it can be seen that the four primer probe sets of this example all have no cross reaction with the common pathogens in the respiratory tract, and no false positive result is obtained, and the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set all have strong specificity.
Example 5 anti-tamper ability verification
Preparing a reaction system shown in table 2 by using four primer probe groups in example 1 and setting preset conditions shown in table 3, and verifying whether the PCR amplification process of the primer probe composition is inhibited by common interfering substances in the respiratory tract; the verification concentration of the interfering substance is the highest theoretically possible concentration in the sample, the Ct value before and after the interfering substance is added is compared by using t test, the significance probability (P value) is calculated, if the analysis result P is more than 0.05, it is indicated that the Ct value before and after the interfering substance is added has no significance difference, that is, the interfering substance has no interference on the detection result, and the verification conditions and the verification result are shown in the following table.
TABLE 16 results of verification of anti-interference ability of primer probe compositions of this example
The verification results in table 16 show that the calculated P values are all greater than 0.05, which means that the Ct values are not significantly changed before and after the interfering substances are added into the sample, and indicate that the PCR amplification reaction of the primer probe composition of the embodiment is not inhibited by the common interfering substances in the respiratory tract, and the primer probe composition of the application has good anti-interference capability.
It should be noted that, in the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described as different from other embodiments, and identical and similar parts between the embodiments are all enough to be referred to each other.
While the application has been described with respect to certain embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the application, and it is intended to cover the application as defined by the appended claims.
Claims (11)
1. A primer probe composition comprising at least one of a first primer probe set for detecting a novel coronavirus N gene and a second primer probe set for detecting a novel coronavirus ORF1ab gene;
The first primer probe group comprises an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2 and a probe shown as SEQ ID NO. 3;
the second primer probe group comprises an upstream primer shown as SEQ ID NO.4, a downstream primer shown as SEQ ID NO.5 and a probe shown as SEQ ID NO. 6;
the primer probe composition also comprises a third primer probe group for detecting influenza A virus, wherein the third primer probe group comprises an upstream primer shown as SEQ ID NO.7, a downstream primer shown as SEQ ID NO.8 and a probe shown as SEQ ID NO. 9.
2. The primer probe composition of claim 1, wherein the primer probe composition further comprises a fourth primer probe set for detecting influenza B virus,
the fourth primer probe group comprises an upstream primer shown as SEQ ID NO.10, a downstream primer shown as SEQ ID NO.11 and a probe shown as SEQ ID NO. 12.
3. The primer probe composition according to any one of claims 1 to 2, wherein the first primer probe set probe, the second primer probe set probe, the third primer probe set probe and the fourth primer probe set probe are labeled with modification groups exhibiting different fluorescent colors, respectively.
4. The primer probe composition of claim 3, wherein the modifying group comprises a fluorescent group and a quenching group;
the fluorescent group comprises at least one of a Cy5 fluorescent group, a VIC fluorescent group, a FAM fluorescent group and a ROX fluorescent group;
the quenching group comprises at least one of a TAMRA quenching group, an MGB quenching group, a BHQ1 quenching group and a BHQ2 quenching group.
5. A kit comprising qPCR premix, reverse transcriptase and the primer probe composition of any one of claims 1 to 4.
6. The kit of claim 5, wherein the kit satisfies at least one of the following characteristics:
at least one of the upstream primer, the downstream primer and the probe of the first primer probe group has the concentration of 70-150 mug/mL;
at least one of the upstream primer, the downstream primer and the probe of the second primer probe set has a concentration of 70-150 mug/mL;
at least one of the upstream primer, the downstream primer and the probe of the third primer probe group has the concentration of 70-150 mug/mL;
at least one of the upstream primer, the downstream primer and the probe of the fourth primer probe set has a concentration of 70-150 mug/mL.
7. Use of a primer probe composition according to any one of claims 1-4 or a kit according to any one of claims 5-6 for detecting novel coronaviruses and influenza a viruses.
8. Use of a primer probe composition according to any one of claims 1-4 or a kit according to any one of claims 5-6 for detecting at least one of influenza a virus and influenza b virus and a novel coronavirus.
9. The use according to any one of claims 7-8, wherein the primer probe composition or the kit is used in a method for detecting novel coronaviruses and influenza a viruses, wherein the PCR amplification reaction comprises a reverse transcription stage, a pre-denaturation stage, a denaturation stage and an annealing stage, and wherein the pre-set conditions of the PCR amplification reaction comprise at least one of the following conditions:
the preset reverse transcription temperature of the reverse transcription stage is 40-50 ℃;
the preset pre-denaturation temperature of the pre-denaturation stage is 90-97 ℃;
the preset denaturation temperature of the denaturation stage is 90-97 ℃;
the preset annealing temperature of the annealing stage is 45-60 ℃;
the preset cycle number of the PCR amplification reaction is 30-50.
10. The use according to any one of claims 7 to 8, wherein the kit comprises a reverse transcriptase, and wherein the kit is used in a method for detecting novel coronaviruses and influenza a viruses, and wherein the reverse transcriptase is used in an amount of 15U to 40U in a single detection procedure.
11. The use according to any one of claims 7-8, wherein the use of the primer probe composition or the kit satisfies at least one of the following characteristics:
the lowest detection limit of the primer probe composition or the kit against the novel coronavirus is 15-150 copies/mL;
the lowest detection limit of the primer probe composition or the kit against influenza A virus is 30-150 copies/mL;
the lowest detection limit of the primer probe composition or the kit against the influenza B virus is 150-750 copies/mL.
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