CN111349720A - Nucleic acid reagent, kit, system and method for detecting respiratory tract infection virus - Google Patents
Nucleic acid reagent, kit, system and method for detecting respiratory tract infection virus Download PDFInfo
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Abstract
The present disclosure relates to a nucleic acid reagent for detecting respiratory tract infection virus, wherein the nucleic acid reagent comprises primers represented by SEQ ID NO.1 to 8 and probes represented by SEQ ID NO.11 to 14, which are stored independently of each other or randomly mixed with each other. The nucleic acid reagent, the kit, the system and the method for detecting the novel coronavirus, the influenza A virus and the influenza B virus are established through the primers and the probes, and meanwhile, an internal reference gene is added as a reference, so that the rapid, comprehensive, sensitive, specific and automatic detection result judgment is realized, and the sensitivity, the specificity and the simplicity of simultaneously detecting the detection targets are obviously improved.
Description
Technical Field
The present disclosure relates to the field of biotechnology, and in particular, to a nucleic acid reagent, a kit, a system and a method for detecting respiratory tract infection viruses.
Background
Respiratory Tract Infection (RTI) is a common disease that occurs widely in people of any age, sex and region, and poses a serious disease burden on individuals, families and society. Viral infection is an important factor causing respiratory tract infection, and acute viral respiratory tract infection is the leading factor leading to hospitalization of infants and children in developed countries; in developing countries, acute viral respiratory infections are the leading cause of death in infants and children.
The novel coronavirus is a respiratory tract infection virus discovered for the first time in the end of 2019, is named as 2019-nCoV, can cause severe pneumonia symptoms, and can cause death of patients when severe. Therefore, the diagnosis of the novel coronavirus infection and the common respiratory virus infection is carried out early, and the isolation and treatment of the patient infected by the novel coronavirus are carried out early, so that the diagnosis and treatment method is very important for restraining the development of epidemic situations and relieving the social pressure.
The isolated culture of the virus is a common method for detecting the respiratory tract infection virus, the detection result is often used as a 'gold standard' for the accurate diagnosis of the respiratory tract infection virus, the common cultured cells comprise human lung cancer cells (A59), Madin Darby canine kidney cells (MDCK) and the like, the culture time of different viruses is different according to different culture samples, the culture time is generally different from several days to two weeks, the culture time is longer, the rapid detection of the respiratory tract virus infection is not facilitated, the delay of treatment is easy to cause, and the specificity and the sensitivity of the method are poorer, so the method is not suitable for early diagnosis when the infection degree is lighter.
Disclosure of Invention
The purpose of the disclosure is to provide a nucleic acid reagent, a kit and a system for quickly and accurately detecting respiratory tract infection viruses, which have high specificity and sensitivity.
In order to achieve the above objects, the present disclosure provides a nucleic acid reagent for detecting respiratory tract infection virus, wherein the nucleic acid reagent comprises primers represented by seq id nos. 1 to 8 and probes represented by seq id nos. 11 to 14, which are stored independently of each other or randomly mixed with each other, respectively.
Alternatively, in the nucleic acid reagent, the content of the primers represented by SEQ ID Nos. 1 to 8 is 0.4 to 0.6. mu. mol, respectively, and the content of the probes represented by SEQ ID Nos. 11 to 14 is 0.1 to 0.3. mu. mol, respectively.
Optionally, the nucleic acid reagents further comprise an internal standard control;
the internal standard control contains primers shown by SEQIDNO.9-10 and probes shown by SEQIDNO.15, wherein the content of the primers respectively shown by SEQIDNO.9-10 is 0.2-0.4 mu mol, and the content of the probes shown by SEQIDNO.15 is 0.1-0.3 mu mol.
Optionally, the nucleic acid reagent comprises tube a and tube B; the tube A contains primers shown by SEQ ID NO.1-4 and 9-10 and probes shown by SEQ ID NO.11-12 and 15; the tube B contains primers shown by SEQ ID NO.5-8 and 9-10 and probes shown by SEQ ID NO.13-14 and 15.
Alternatively, the probes shown in SEQ ID NO.11 and 13 have a first fluorescent label; the probes shown as SEQ ID NO.12 and 14 have a second fluorescent label; the probe shown as SEQ ID NO.15 has a third fluorescent label; the first fluorescent label, the second fluorescent label and the third fluorescent label are each different and are each independently selected from one of a FAM fluorescent label, a JOE fluorescent label, a CY5 fluorescent label, a VIC fluorescent label and a ROX fluorescent label.
Optionally, the respiratory infection virus comprises at least one of a novel coronavirus, an influenza a virus, and an influenza b virus.
The present disclosure also provides a kit for detecting a respiratory infection virus, the kit comprising the nucleic acid reagent according to any one of the above, and optionally, the kit further comprises at least one of a reaction system buffer, a reverse transcriptase, a DNA polymerase, a positive control, and water.
The present disclosure also provides a use of the nucleic acid reagent of any one of the above in preparing a kit for detecting respiratory infectious virus.
The present disclosure also provides a system for detecting respiratory tract infection viruses, the system comprising a PCR instrument having an a-tube detector and a B-tube detector, a computing device, and an output device; the tube A detector and the tube B detector are nucleic acid reagent storage containers loaded with any one of the nucleic acid reagents; the PCR instrument comprises a first fluorescence channel, a second fluorescence channel and a third fluorescence channel; the first, second, and third fluorescent channels are each different and each independently selected from one of a FAM fluorescent channel, a JOE fluorescent channel, and a CY5 fluorescent channel; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the blank control, the positive control and the internal standard control are established, the detection result is effective;
if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 38, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 38 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative;
if the detection results of the first fluorescence channel and the second fluorescence channel of the tube A are positive, judging that the sample contains the novel coronavirus; if the detection result of the third fluorescence channel of the tube A is positive, judging that the sample is normally extracted and the detection result is normal;
if the detection result of the first fluorescence channel of the tube B is positive, the sample is judged to contain influenza A virus; if the detection result of the second fluorescent channel of the tube B is positive, judging that the sample contains influenza B virus; if the detection result of the third fluorescence channel of the tube B is positive, the sample is judged to be normally extracted, and the detection result is normal.
The present disclosure also provides a method for detecting a respiratory infection virus, wherein the method comprises: carrying out PCR amplification on a nucleic acid sequence of a sample to be detected by adopting the nucleic acid reagent; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel and a third fluorescence channel; the first, second, and third fluorescent channels are distinct and each independently selected from one of the FAM, JOE, and CY5 fluorescent channels; and the following discrimination is made:
if the blank control, the positive control and the internal standard control are established, the detection result is effective;
if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 38, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 38 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative;
if the detection results of the first fluorescence channel and the second fluorescence channel of the tube A are positive, judging that the sample contains the novel coronavirus; if the detection result of the third fluorescence channel of the tube A is positive, judging that the sample is normally extracted and the detection result is normal;
if the detection result of the first fluorescence channel of the tube B is positive, the sample is judged to contain influenza A virus; if the detection result of the second fluorescent channel of the tube B is positive, judging that the sample contains influenza B virus; if the detection result of the third fluorescence channel of the tube B is positive, the sample is judged to be normally extracted, and the detection result is normal.
The beneficial effect of this disclosure lies in:
by utilizing the nucleic acid reagent, the kit, the system and the method provided by the disclosure, the detection of the novel coronavirus, the influenza A virus and the influenza B virus in a sample to be detected can be quickly and accurately realized, the complex operation of virus culture is avoided, and the following detection effects are achieved:
higher multiple detection capability
The detection method established by the disclosure can screen novel coronary viruses, influenza A viruses and influenza B viruses in 2 reaction systems simultaneously, quickly and simply identify the types of pathogens causing respiratory tract infection, and save time, labor and reagent cost.
(II) high sensitivity
The detection method established by the disclosure can realize the simultaneous detection of novel coronavirus, influenza A virus and influenza B virus, the detection sensitivity of each target gene for detecting target pathogen in a reaction system can reach 500copies/mL, and the detection sensitivity is high.
(III) high specificity
In the detection method established by the disclosure, all primers and probes are subjected to BLAST comparison analysis, have high conservation and specificity, can distinguish detection targets from each other, and can also distinguish other respiratory tract infection pathogens which are similar in species, identical in living environment, homologous in nucleic acid sequence, easy to cause the same or similar clinical symptoms, and normal parasitism or easy to be complicated at a sampling part, wherein the respiratory tract infection pathogens at least comprise respiratory syncytial virus, adenovirus, parainfluenza virus, coronavirus NL63, coronavirus OC43, coronavirus HKUI, coronavirus 229E, middle east respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus, rhinovirus, bocavirus, measles virus, enterovirus, cytomegalovirus, haemophilus influenzae, staphylococcus aureus, staphylococcus epidermidis and staphylococcus aureus, Streptococcus pneumoniae, neisseria meningitidis, bordetella pertussis and the like, and therefore, the detection method established by the disclosure has high specificity and can accurately distinguish a detection target from a non-detection target.
(IV) prevention of false negative results
In the detection method established by the disclosure, false negative detection results can be effectively prompted by using internal standard control.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, and are not intended to limit the present disclosure.
The first aspect of the present disclosure provides a nucleic acid reagent for detecting respiratory tract infection virus, wherein the nucleic acid reagent comprises primers represented by SEQ ID NO.1-8 and probes represented by SEQ ID NO.11-14, each of which is stored independently of each other or in any mixture with each other.
The nucleic acid reagent provided by the disclosure can rapidly and accurately screen and identify the novel coronavirus, the influenza A virus and the influenza B virus by adopting a multiple fluorescence PCR technology. The nucleic acid reagent has good specificity and high sensitivity for detecting the 3 respiratory tract infection viruses, and can be used for early diagnosis of patients with pneumonia symptoms.
In the multiplex fluorescence PCR technology, the combination effect of the probe and the primer has an important influence on the amplification effect, the primer and the probe not only consider the problem of co-amplification of the primer and the probe of different target genes in a reaction system in the design process, namely, the conditions of evaluating the consistency of Tm values and the homogenization of GC content, avoiding the occurrence of hairpin structures, primer dimers and the like, but also ensure that the alternative primer and the probe segment can respectively and comprehensively cover the 3 respiratory tract infection viruses, and the primers and the probe have good specificity, high coverage and high sensitivity.
In accordance with the present disclosure, the relative amounts of each primer and/or probe in the above-described nucleic acid reagents can vary over a wide range. For example, in the nucleic acid reagent, the primers represented by SEQ ID Nos. 1 to 8 are contained in an amount of 0.4 to 0.6. mu. mol, respectively, and the probes represented by SEQ ID Nos. 11 to 14 are contained in an amount of 0.1 to 0.3. mu. mol, respectively.
According to the disclosure, the nucleic acid reagent may further include an internal standard control in order to control the internal quality of the reaction system, better judge whether the reaction is interfered, and exclude false negative results. Further, the internal standard control may contain primers represented by SEQ ID NO.9 to 10 and probes represented by SEQ ID NO.15, in which case, the contents of the primers represented by SEQ ID NO.9 to 10 are respectively 0.2 to 0.4. mu. mol, and the contents of the probes represented by SEQ ID NO.15 are respectively 0.1 to 0.3. mu. mol. The internal standard control can effectively prompt false negative detection results caused by misoperation, PCR inhibitors and the like on one hand, and can be used for judging the quality of the sample on the other hand, so that the detection is carried out at the same sample level, and the phenomenon that the quantitative results of the respiratory tract infection viruses are greatly different due to inconsistent sample consistencies is prevented. The internal standard control primer and the probe can be designed by taking endogenous beta-actin internal standard genes as templates.
According to the present disclosure, in order to enhance the accuracy of the detection result, the nucleic acid reagent may be stored separately, for example, the nucleic acid reagent may include a tube a and a tube B; the tube A can contain primers shown by SEQ ID NO.1-4 and 9-10 and probes shown by SEQ ID NO.11-12 and 15; the tube B can contain primers shown by SEQ ID NO.5-8 and 9-10 and probes shown by SEQ ID NO.13-14 and 15.
Further, in order to allow amplification of different probes in the same system to be recognized separately, the probes may be aligned and labeled with fluorescence. For example, as one embodiment, the probes shown in seq id nos. 11 and 13 can have a first fluorescent label; the probes shown in seq id nos. 12 and 14 may have a second fluorescent label; the probe shown as SEQ ID No.15 can have a third fluorescent label; the first fluorescent label, the second fluorescent label and the third fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a JOE fluorescent label, a CY5 fluorescent label, a VIC fluorescent label and a ROX fluorescent label. As a particularly preferred embodiment, the probes shown in SEQ ID Nos. 11 and 13 have FAM fluorescent labels; the probes shown as SEQ ID NO.12 and 14 have JOE fluorescent labels; the probe shown as SEQ ID NO.15 has a CY5 fluorescent label.
According to the present disclosure, the respiratory infection virus includes at least one of a novel coronavirus, an influenza a virus, and an influenza b virus. Preferably, the target gene of the novel coronavirus may be an ORF1ab gene and an N gene, the target gene of the influenza a virus may be an M2 gene, and the target gene of the influenza b virus may be an NS1 gene.
The second aspect of the present disclosure also provides a kit for detecting a respiratory infection virus, the kit comprising the nucleic acid reagent of any one of the above, and optionally, the kit further comprises at least one of a reaction system buffer, a reverse transcriptase, a DNA polymerase, a positive control, and water.
The kit disclosed by the invention can realize rapid, comprehensive, accurate, sensitive, specific and automatic detection result judgment, and obviously improves the sensitivity, specificity and simplicity of simultaneously detecting the novel coronavirus, the influenza A virus and the influenza B virus.
The third aspect of the present disclosure also provides a use of the nucleic acid reagent of any one of the above in the preparation of a kit for detecting a respiratory infection virus.
The fourth aspect of the present disclosure also provides a system for detecting respiratory tract infection viruses, the system comprising a PCR instrument having an a-tube detector and a B-tube detector, a computing device, and an output device; the tube A detector and the tube B detector are nucleic acid reagent storage containers loaded with any one of the nucleic acid reagents; the PCR instrument comprises a first fluorescence channel, a second fluorescence channel and a third fluorescence channel; the first, second, and third fluorescent channels are distinct and each independently selected from one of the FAM, JOE, and CY5 fluorescent channels; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the blank control, the positive control and the internal standard control are established, the detection result is effective;
if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 38, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 38 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative;
if the detection results of the first fluorescence channel and the second fluorescence channel of the tube A are positive, judging that the sample contains the novel coronavirus; if the detection result of the third fluorescence channel of the tube A is positive, judging that the sample is normally extracted and the detection result is normal;
if the detection result of the first fluorescence channel of the tube B is positive, the sample is judged to contain influenza A virus; if the detection result of the second fluorescent channel of the tube B is positive, judging that the sample contains influenza B virus; if the detection result of the third fluorescence channel of the tube B is positive, the sample is judged to be normally extracted, and the detection result is normal.
The fifth aspect of the present disclosure also provides a method for detecting a respiratory tract infection virus, wherein the method comprises: carrying out PCR amplification on a nucleic acid sequence of a sample to be detected by adopting the nucleic acid reagent; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel and a third fluorescence channel; the first, second, and third fluorescent channels are distinct and each is independently selected from one of the FAM, JOE, and CY5 fluorescent channels; and the following discrimination is made:
if the blank control, the positive control and the internal standard control are established, the detection result is effective;
if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 38, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 38 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative;
if the detection results of the first fluorescence channel and the second fluorescence channel of the tube A are positive, judging that the sample contains the novel coronavirus; if the detection result of the third fluorescence channel of the tube A is positive, judging that the sample is normally extracted and the detection result is normal;
if the detection result of the first fluorescence channel of the tube B is positive, the sample is judged to contain influenza A virus; if the detection result of the second fluorescent channel of the tube B is positive, judging that the sample contains influenza B virus; if the detection result of the third fluorescence channel of the tube B is positive, the sample is judged to be normally extracted, and the detection result is normal.
Wherein, the PCR amplification condition can be as follows:
a：45℃10min；
b：95℃5min；
c：95℃ 15s；
d: 15-60s at 60-70 ℃, and 35-45 reactions are circulated c-d. The reaction conditions include two different annealing temperatures, which can improve the sensitivity of the detection method.
The sample to be tested can be pharyngeal swab and sputum of a clinical patient. The sample to be detected can be processed by a boiling method or a commercial kit to obtain the RNA to be detected.
Preferably, the method for detecting respiratory tract infection virus of the present disclosure is not used for diagnosis, or qualitative and quantitative results of the novel coronavirus, influenza a virus and influenza b virus do not have a one-to-one correlation with whether a disease occurs, and do not belong to a diagnosis result, but qualitative and quantitative detection structures of the novel coronavirus, influenza a virus and influenza b virus can be used as intermediate information for reference of a clinician.
The method disclosed by the invention can quickly, sensitively and specifically realize the systematic screening of the novel coronavirus, the influenza A virus and the influenza B virus, the detection process is simple, the result is automatically interpreted and reliable, and the time, the labor and the reagent cost are saved.
The present disclosure is further illustrated in detail below by way of examples, but the present disclosure is not limited thereto. The starting materials, reagents, instruments and equipment referred to in the following examples were all available commercially except as specifically indicated.
Example 1 detection method and determination of detection result
1. Primer and probe synthesis
Sequence synthesis was performed according to the primer sequences shown in Table 1 and the probe sequences shown in Table 2. Y in the sequence represents degenerate base T/C; r represents a degenerate base A/G; w represents a degenerate base A/T; s represents a degenerate base C/G. In the probe, FAM is 6-carboxyfluorescein, JOE is 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein, CY5 is 5H-indocyanine, and BQ1 and BQ3 are quenching groups.
TABLE 1
TABLE 2
2. Sample processing
Extracting the throat swab or the liquefied sputum by using a commercial kit according to an instruction, and taking the extracted nucleic acid as a PCR amplification template; or extracting throat swab or liquefied sputum by boiling method, centrifuging for several times, and collecting supernatant as PCR amplification template.
3. Construction of a detection System
The reaction system is prepared by the following steps of preparing 5 × PCRbuffer5 mu L, 25 × HotstadrDNA polymerase 1 mu L, reverse transcriptase 1 mu L, 10 × primer probe mixture 2.5 mu L, RNA template 5 mu L and ultrapure water to 25 mu L, wherein the specific composition of the 5 × PCRbuffer comprises 100mM Tris-HCl (pH8.3), 100 KCl100mM, Tween-200.2%, dNTP5mM, MgCl220mM, 25 × concentration of Hotsta rDNA polymerase 2U/. mu.L, concentration of reverse transcriptaseThe degree is 2U/mu L; the final concentration of each primer in the reaction system is 0.3-0.6. mu.M, and the final concentration of each probe is 0.1-0.3. mu.M.
The kit is divided into a tube A and a tube B, wherein the tube A contains primers shown by SEQ ID NO.1-4 and 9-10 in the table 1 and probes shown by SEQ ID NO.11-12 and 15 in the table 2; tube B contains the primers shown by SEQ ID Nos. 5-8 and 9-10 in Table 1 above and the probes shown by SEQ ID Nos. 13-14 and 15 in Table 2 above.
Placing the PCR tube into a fluorescent quantitative PCR instrument, selecting FAM, JOE and CY5 as a report group, and carrying out the following reaction procedures:
a：45℃10min；
b：95℃5min；
c：95℃15s；
d: 15-60s at 60-70 ℃, and 35-45 reactions are circulated c-d.
And (3) judging a detection result:
if the blank control, the positive control and the internal standard control are established, the detection result is effective;
if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 38, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 38 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative; if the fluorescent channel does not detect the S-type amplification curve but reports the CT value, it is determined that there is non-specific amplification in the detection result.
If the detection results of the A tube FAM fluorescence channel and the JOE fluorescence channel are positive, determining that the sample contains the novel coronavirus; if the detection result of the tube A CY5 fluorescence channel is positive, judging that the sample extraction is normal and the detection result is normal;
if the detection result of the tube B FAM fluorescent channel is positive, judging that the sample contains influenza A virus; if the detection result of the B tube JOE fluorescence channel is positive, judging that the sample contains influenza B virus; if the detection result of the tube B CY5 fluorescence channel is positive, the sample extraction is judged to be normal, and the detection result is normal.
Example 2 minimum detection Limit validation
Test samples for evaluation: total RNAs of the novel coronavirus, influenza A virus and influenza B virus were extracted using a commercial kit, and the target gene concentration was quantified to 105After copying/mL, the samples were diluted in a gradient to give templates for evaluation at concentrations of 50000 copies/mL, 5000 copies/mL and 500copies/mL, respectively.
The template for evaluation of each concentration was examined according to the examination method of example 1, 20 times for each concentration gradient, and the average was taken as the final examination result, as shown in Table 3.
TABLE 3
Note: "+" indicates positive, and n/20 indicates a detection rate of 20 repeated n detections
As can be seen from Table 3, the lowest detection limit concentration of the kit for detecting the target genes of the novel coronavirus, the influenza A virus and the influenza B virus reaches 500copies/mL, and the kit has high detection sensitivity.
Example 3 specificity verification
Respiratory syncytial virus (from institute of pathogenic biology, institute of Chinese medical science, code 202002001), adenovirus (from institute of pathogenic biology, institute of Chinese medical science, code 202002002), parainfluenza virus (from institute of pathogenic biology, institute of Chinese medical science, code 202002003), coronavirus NL63 (from institute of pathogenic biology, institute of Chinese medical science, code 202002004), coronavirus OC43 (from institute of pathogenic biology, institute of Chinese medical science, code 202002005), coronavirus HKUI (from institute of pathogenic biology, institute of Chinese medical science, code 202002006), coronavirus 229E (from institute of pathogenic biology, code 202002007), and respiratory syndrome coronavirus HKUI (in vitro transcribed RNA, from institute of pathogenic biology, institute of Chinese medical science, code 202002007), 202002008), Severe acute respiratory syndrome coronavirus (in vitro transcribed RNA, from institute of pathogenic biology of Chinese medical science institute, 202002009), rhinovirus (from institute of pathogenic biology of Chinese medical science institute, 202002010), bocavirus (from institute of pathogenic biology of Chinese medical science institute, 202002011), measles virus (from institute of pathogenic biology of Chinese medical science institute, 202002012), enterovirus (from institute of pathogenic biology of Chinese medical science institute, 202002013), cytomegalovirus (from institute of pathogenic biology of Chinese medical science institute, 202002014), Haemophilus influenzae (from institute of pathogenic biology of Chinese medical science institute, 202002015), Staphylococcus aureus (from institute of pathogenic biology of Chinese medical science institute, 202002016), Staphylococcus epidermidis (from institute of pathogenic biology of Chinese academy of medicine, 202002017), Streptococcus pneumoniae (from institute of pathogenic biology of Chinese academy of medicine, 202002018), Neisseria meningitidis (from institute of pathogenic biology of Chinese academy of medicine, 202002019), Bordetella pertussis (from institute of pathogenic biology of Chinese academy of medicine, 202002020), and other pathogens causing respiratory tract infection were used as specificity evaluation samples.
By applying the kit provided by the disclosure and detecting the specificity evaluation sample according to the detection method of the embodiment 1, under the condition that negative control, positive control and positive internal control are all satisfied, non-specific fluorescent signals do not appear on the target to be detected, which shows that the kit provided by the disclosure can effectively distinguish non-detection target pathogens and has better specificity.
EXAMPLE 4 shelf-Life testing of kits
A mixed template of a novel coronavirus, an influenza A virus and an influenza B virus each having a target gene concentration of 500copies/mL was used as a test sample for evaluation, and the test samples were frozen in 9 portions at day 0 in a refrigerator at-70 ℃. And (3) storing the assembled kit at the temperature of-20 ℃, and performing storage period tests on the kit with the time periods of 0, 10, 15, 30, 60, 90, 120, 150, 180 and 360 days respectively.
The results show that the kit disclosed by the invention is stored in a refrigerator at the temperature of-20 ℃, and the detection is positive in different storage periods, which indicates that the storage period of the kit is at least 360 days.
Example 5 coverage verification
30 throat swab samples or sputum samples for evaluation were selected as samples for coverage verification.
By using the kit provided by the disclosure, the sample for coverage verification is detected according to the detection method of the embodiment 1, and under the condition that the negative control, the positive control and the positive internal control are all satisfied, the novel coronavirus, the influenza a virus and the influenza b virus in the sample for coverage verification are all detected, which shows that the kit disclosed by the disclosure has good coverage.
Comparative example
1. Primer and probe synthesis
Sequence synthesis was performed according to the primer and probe sequences shown in tables 4 and 5.
TABLE 4
TABLE 5
2. Minimum detection limit verification
The minimum detection limit verification was performed according to the method of example 2, and the detection results are shown in Table 6.
TABLE 6
Note: "+" indicates positive, and n/20 indicates a detection rate of 20 repeated n detections
As can be seen from Table 6, for trace amounts of target genes of the novel coronavirus, the influenza A virus and the influenza B virus in the sample, the detection rate of the novel coronavirus, the detection rate of the influenza A virus and the detection rate of the influenza B virus of the primers and the probes in the comparative examples are 60%, 25% and 40% respectively at the target gene concentration of 500copies/mL of the target bacteria. Indicating that the disclosed kit has greater detection capability than the comparative example.
3. Specificity verification
Specificity verification was performed according to the method of example 3. The results show that the primers and probes of the comparative examples amplify SARS virus, rhinovirus, coronavirus NL63 and coronavirus 229E non-specifically. It is demonstrated that the kit of the present disclosure has better detection specificity compared to the comparative example.
As can be seen from the examples and comparative examples, the method can quantitatively detect the novel coronavirus, the influenza A virus and the influenza B virus at one time, and has the advantages of low minimum detection limit, high sensitivity and high specificity.
The preferred embodiments of the present disclosure have been described above in detail, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications are within the protective scope of the present disclosure.
It should be noted that the respective technical features described in the above embodiments may be combined in any suitable manner, for example, the a-tube detection target and the B-tube detection target may be interchanged. The disclosure is not further described in terms of the various possible combinations to avoid unnecessary repetition.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Sequence listing
<110> institute of pathogenic biology of Chinese academy of medical sciences
BEIJING APPLIED BIOLOGICAL TECHNOLOGIES Co.,Ltd.
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Claims (10)
1. A nucleic acid reagent for detecting respiratory tract infection virus, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-8 and probes shown in SEQ ID NO.11-14, which are respectively stored independently of each other or randomly mixed with each other.
2. The nucleic acid reagent according to claim 1, wherein the content of each of the primers represented by SEQ ID nos. 1 to 8 and the content of each of the probes represented by SEQ ID nos. 11 to 14 in the nucleic acid reagent are 0.4 to 0.6. mu. mol and 0.1 to 0.3. mu. mol, respectively.
3. The nucleic acid reagent of claim 1, wherein the nucleic acid reagent further comprises an internal standard control;
the internal standard control contains primers shown by SEQ ID NO.9-10 and probes shown by SEQ ID NO.15, wherein the content of the primers shown by SEQ ID NO.9-10 is 0.2-0.4 mu mol respectively, and the content of the probes shown by SEQ ID NO.15 is 0.1-0.3 mu mol.
4. The nucleic acid reagent of claim 3, wherein the nucleic acid reagent comprises an A-tube and a B-tube; the tube A contains primers shown in SEQ ID NO.1-4 and 9-10 and probes shown in SEQ ID NO.11-12 and 15; the tube B contains primers shown in SEQ ID NO.5-8 and 9-10 and probes shown in SEQ ID NO.13-14 and 15.
5. The nucleic acid reagent according to claim 4, wherein the probes represented by SEQ ID No.11 and 13 have a first fluorescent label; the probes shown in SEQ ID NO.12 and 14 have a second fluorescent label; the probe shown in SEQ ID NO.15 has a third fluorescent label; the first fluorescent label, the second fluorescent label and the third fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a JOE fluorescent label, a CY5 fluorescent label, a VIC fluorescent label and a ROX fluorescent label.
6. The nucleic acid agent according to any one of claims 1 to 5, wherein the respiratory tract infection virus comprises at least one of a novel coronavirus, an influenza A virus and an influenza B virus.
7. A kit for detecting a respiratory infection virus, the kit comprising the nucleic acid reagent according to any one of claims 1 to 6, and optionally, the kit further comprises at least one of a reaction system buffer, a reverse transcriptase, a DNA polymerase, a positive control and water.
8. Use of the nucleic acid reagent of any one of claims 1 to 6 in the preparation of a kit for detecting respiratory tract infection virus.
9. A system for detecting respiratory tract infection viruses, the system comprising a PCR instrument having an a-tube detector and a B-tube detector, a computing device and an output device; the tube A detector and the tube B detector are respectively a nucleic acid reagent storage container loaded with the nucleic acid reagent of any one of claims 4 to 6; the PCR instrument comprises a first fluorescence channel, a second fluorescence channel and a third fluorescence channel; the first, second, and third fluorescent channels are distinct and each independently selected from one of the FAM, JOE, and CY5 fluorescent channels; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the blank control, the positive control and the internal standard control are established, the detection result is effective;
if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 38, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 38 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative;
if the detection results of the first fluorescence channel and the second fluorescence channel of the tube A are positive, judging that the sample contains the novel coronavirus; if the detection result of the third fluorescence channel of the tube A is positive, judging that the sample is normally extracted and the detection result is normal;
if the detection result of the first fluorescence channel of the tube B is positive, judging that the sample contains influenza A virus; if the detection result of the second fluorescent channel of the tube B is positive, judging that the sample contains the influenza B virus; and if the detection result of the third fluorescence channel of the tube B is positive, judging that the sample is normally extracted and the detection result is normal.
10. A method for detecting a respiratory infection virus, wherein the method comprises: performing PCR amplification on a nucleic acid sequence of a sample to be detected by using the nucleic acid reagent according to any one of claims 4 to 6; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel and a third fluorescence channel; the first, second, and third fluorescent channels are distinct and each independently selected from one of the FAM, JOE, and CY5 fluorescent channels; and the following discrimination is made:
if the blank control, the positive control and the internal standard control are established, the detection result is effective;
if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 38, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 38 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative;
if the detection results of the first fluorescence channel and the second fluorescence channel of the tube A are positive, judging that the sample contains the novel coronavirus; if the detection result of the third fluorescence channel of the tube A is positive, judging that the sample is normally extracted and the detection result is normal;
if the detection result of the first fluorescence channel of the tube B is positive, judging that the sample contains influenza A virus; if the detection result of the second fluorescent channel of the tube B is positive, judging that the sample contains the influenza B virus; and if the detection result of the third fluorescence channel of the tube B is positive, judging that the sample is normally extracted and the detection result is normal.
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| CN112063756A (en) * | 2020-09-17 | 2020-12-11 | 中山大学达安基因股份有限公司 | Method and kit for multiplex detection of respiratory virus nucleic acid |
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