CN116411133A - Compositions, methods and uses for novel coronavirus and monkey pox virus joint detection - Google Patents
Compositions, methods and uses for novel coronavirus and monkey pox virus joint detection Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to a novel joint inspection composition, a method and application of coronavirus 2019-nCoV and monkey pox virus. The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting different sites on different pathogens, so that the detection of novel coronaviruses and monkey pox viruses is realized simultaneously in a single-tube reaction system. So that different pathogens can be treated differently, thereby making the treatment and prevention more effective. Meanwhile, the composition provided by the invention has higher detection sensitivity, reaches 200 copies/mL, and is more accurate in detection.
Description
Technical Field
The invention belongs to the field of molecular biology detection, and particularly relates to a novel joint inspection composition, a method and application of coronavirus 2019-nCoV and monkey pox virus.
Background
Infection by the novel coronavirus (2019-nCoV) is widely spread worldwide, causing serious effects to the world, and is listed as an "international sudden public health event" by the world health organization. Based on epidemiological investigation, the novel coronavirus has a latency period of 1-14 days, and symptoms mainly include fever, hypodynamia, dry cough, severe patients show dyspnea, sepsis shock, coagulation dysfunction, multiple organ failure, metabolic acidosis which is difficult to correct, and the like.
The monkey poxvirus is a double-stranded DNA virus, belongs to zoonosis, is first found on experimental macaques of Copenhagen of Danish in 1958, and is first reported in Zaele area in 1970 to appear in infection of mankind poxvirus. The monkey poxvirus belongs to the family of poxviridae and has similar symptoms of infection to smallpox, such as fever, cough, lymphadenectasis and general pain, and can be possibly fatal by directly contacting a patient or infected animals or by transmitting the virus through body fluid and respiratory droplets of the patient. More than 100 cases have been diagnosed in less than one month since the first case of month 5 of 2022, WHO expanded monitoring in non-endemic countries, and current information suggests that interpersonal transmission is occurring in people in intimate physical contact with symptomatic cases, and now more and more people are no longer immune to the monkey poxvirus.
One clinical manifestation may be caused by multiple pathogens, presenting difficulties for early clinical diagnosis, and different viruses may be treated differently in their clinical treatments. For easily infected viruses, such as novel coronaviruses, monkey pox, etc., early detection and early treatment are required, and the infectious sources are controlled in time to block the transmission path. There is a need in the art to distinguish between novel coronavirus and monkey poxvirus infections with similar symptoms, provide guidance for subsequent control, treatment, and targeted action, and thus there is a need in the art for a related product that can detect and distinguish between novel coronaviruses and monkey poxviruses simultaneously.
Disclosure of Invention
In view of this, the present invention provides in a first aspect a novel composition for joint inspection of coronaviruses and monkey poxviruses, comprising:
an upstream primer and a downstream primer and a probe for detecting 2019-nCoV ORF1ab genes as shown in SEQ ID NO. 1-3;
an upstream primer and a downstream primer and a probe for detecting 2019-nCoV N gene as shown in SEQ ID NO. 4-6; and
the upstream and downstream primers and probes for detecting the monkey poxvirus are shown as SEQ ID NO. 7-9.
In some specific embodiments, the composition further comprises:
the upstream and downstream primers and probes for detecting the monkey poxvirus are shown as SEQ ID NO. 10-12.
The use of additional primers and probes for the monkey poxvirus can be further pre-leak detected. The detection failure caused by the mutation of the first target gene is avoided, the monkey poxvirus can still be detected, and the detection accuracy is further improved. The probability of false negatives is reduced.
Further, the composition comprises: and detecting the upstream and downstream primers and probes of the internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is a human housekeeping gene.
In some embodiments, the upstream and downstream primers and probes used to detect the internal standard are the internal standard upstream primer shown as SEQ ID NO. 13, the internal standard downstream primer shown as SEQ ID NO. 14, and the internal standard probe shown as SEQ ID NO. 15.
The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting different sites on different pathogens, so that the detection of novel coronaviruses and monkey pox viruses is realized simultaneously in a single-tube reaction system. So that different pathogens can be treated differently, thereby making the treatment and prevention more effective. Meanwhile, the composition provided by the invention has higher detection sensitivity, reaches 200 copies/mL, and is more accurate in detection.
Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.
As used herein, "distinct and non-interfering with each other" means that the fluorophores used for each probe in the composition are different and do not affect each other's detection, i.e., can be performed using different channels. For example FAM, HEX, ROX and CY5 can be used, which groups do not have close absorbance values and can select different channels so as not to interfere with each other.
Further, in some embodiments, the compositions of the present invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to matched upstream and downstream primers and probes that detect a target.
For example, any 4 pairs of the above 5 pairs of primers and probes may be included, any 3 pairs of the above 5 pairs of primers and probes may be included, any 2 pairs of the above 5 pairs of primers and probes may be included, or any 1 pair of the above 5 pairs of primers and probes may be included.
In some specific embodiments, the compositions of the invention are used in fluorescent PCR.
In a specific embodiment, the fluorescent reporter group of the 2019-nCoV ORF1ab gene probe is FAM; the fluorescent reporter group of the 2019-nCoV N gene probe is HEX; the fluorescent reporter group of the monkey poxvirus probe is ROX; the fluorescence reporter group of the internal standard probe is CY5.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' -end of the probe also has a quenching group, such as BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is BHQ1.
Further, the amount of the primer in the composition is 0.2 to 0.5. Mu.M; the amount of probe in the composition is 0.2 to 0.5. Mu.M.
In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.
In a particular embodiment, the ingredients of the composition of the invention are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the invention provides the use of a composition according to the invention as described above for the preparation of a kit for the detection of novel coronaviruses and monkey pox viruses.
In a third aspect, the present invention provides a kit for the joint detection of novel coronaviruses and monkey poxviruses, comprising the composition of the invention as described above.
Further, the kit also comprises a negative quality control and a positive quality control.
In a specific embodiment, the negative quality control is DEPC H 2 O, normal saline and internal standard gene pseudovirus. The positive quality control substance is at least one of fragment plasmid, fragment RNA and pseudovirus of novel coronavirus and monkey pox virus target genes.
Further, the kit also comprises dNTPs, PCR buffer solution and Mg 2+ At least one of them.
Still further, the kit further comprises: at least one of a nucleic acid releasing agent, a nucleic acid extracting agent, a reverse transcriptase and a DNA polymerase.
Further, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTPs, a reverse transcriptase, a DNA polymerase, a PCR buffer solution and Mg 2+ At least one of them.
Further, the concentration of the reverse transcriptase is 5U/reaction to 15U/reaction, for example, the reverse transcriptase may be Neoscript RT reverse transcriptase; the concentration of the DNA polymerase is 3U/reaction-15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a specific embodiment, the kit of the invention comprises: reverse/reverse transcriptase, taq enzyme, mg 2+ 、Mn 2+ Rnasin, dNTPs, primers, probes and PCR buffer.
Common PCR buffer consists of Tris-HCl and MgCl 2 Buffer systems such as KCl and Triton X-100. The total volume in a typical single PCR reaction tube is 20. Mu.l to 200. Mu.l.
In a specific embodiment, the kit of the invention is compatible with digital PCR amplification systems, i.e., can be used directly on a digital PCR instrument for amplification.
In a fourth aspect, a method for joint detection of novel coronaviruses and monkey pox viruses is provided, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
In the present invention, the sample for detection may be a pharyngeal swab, an oropharyngeal swab, blood, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription is carried out at 50-60 ℃ for 3-30 minutes for 1 cycle; cDNA is pre-denatured, the temperature is 95 ℃, the time is 5-40 seconds, and 1 cycle is performed; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
In a specific embodiment, a method for joint inspection of novel coronaviruses and monkey pox viruses for non-diagnostic purposes is provided, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription is carried out at 50-60 ℃ for 3-30 minutes for 1 cycle; cDNA is pre-denatured, the temperature is 95 ℃, the time is 5-40 seconds, and 1 cycle is performed; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
In this context, the term "non-diagnostic purpose" refers to information not intended to obtain whether an individual is infected with a novel coronavirus and a monkey pox virus and is afflicted with pneumonia, monkey pox, and the like. For example, the method may be used to detect the presence or absence of the above-mentioned pathogens in test cultures in experiments aimed at scientific research.
Drawings
FIG. 1 is a graph showing the detection results of the composition of the present invention;
FIGS. 2-4 are graphs showing the sensitivity of the compositions of the present invention;
FIG. 5 is a blank specificity of the composition of the present invention;
FIGS. 6 to 8 are graphs showing the results of the comparative example compositions of the present invention;
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1, primers and probes used in the present invention
TABLE 1
Wherein, the fluorescence reporter group of the 2019-nCoV ORF1ab gene probe is FAM; the fluorescent reporter group of the 2019-nCoV N gene probe is HEX; the fluorescent reporter group of the monkey poxvirus probe is ROX; the fluorescence reporter group of the internal standard probe is CY5.
Example 2 methods for detecting novel coronaviruses and Simian poxviruses
Reverse transcription fluorescence PCR amplification reaction solution: contains 5 XHCV buffer, reverse transcriptase, taq enzyme, mg2+, RNasin, dNTPs, primers, probes, etc. The specific reaction system is shown in Table 2.
TABLE 2
The amplification reaction procedure is shown in Table 3.
TABLE 3 Table 3
Result analysis and judgment:
and after the reaction is finished, automatically storing the result, and respectively analyzing the detection targets and the amplification curves of the internal standard. And (3) adjusting the Start value, end value and Threshold value of Baseline according to the analyzed image (a user can adjust the Start value at 3-15, the End value can be set at 5-20, the amplification curve of the negative control is adjusted to be straight or lower than a Threshold line), clicking Analyze for analysis, so that various parameters meet the requirements in quality control, and recording qualitative results under a Plate window.
Quality control
Negative control: the FAM channel and the HEX/VIC, ROX, CY channel have no Ct value or Ct > 40;
positive control: the Ct of FAM and HEX/VIC, ROX, CY5 channels is less than or equal to 35;
the requirements are met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.
Positive judgment value
The research of the reference value determines that the Ct reference value of the target gene detected by the kit is 40, and the Ct reference value of the internal standard is 40.
Interpretation of test results
Whether an amplification curve exists in the internal standard is analyzed firstly, and if yes, ct is less than or equal to 35, the detection is effective, and the subsequent analysis can be continued:
TABLE 4 Table 4
According to the detection result, the judgment result is as follows:
TABLE 5
If Ct is not detected in the internal standard channel or Ct is more than 40, the content of the cells of the sample to be detected is too low or the inhibition reaction of the interfering substances exists, the detection result of the sample is invalid, the cause is searched and eliminated, and the sample is resampled for repeated tests.
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in example 1 were used to verify the pseudovirus samples of the novel coronaviruses and monkey pox according to the method of example 2, and the results show that the novel coronaviruses and monkey pox can be subjected to joint inspection and distinguished, and the detection results are shown in fig. 1.
Example 4 sensitivity of the composition of the invention
Using the composition of example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate a clinical sample, and multiplex PCR detection was performed on a macrostone fluorescent quantitative PCR instrument. The results are shown in FIGS. 2-4, which show that each channel can still be accurately detected for samples as low as 200 copies/mL, indicating that the sensitivity of the composition of the present invention is 200 copies/mL.
EXAMPLE 5 specificity of the composition of the invention
In order to test the blank specificity of the composition of example 1 of the present invention, a negative control was used as a sample, and the test was performed according to the procedure described above. As shown in FIG. 5, each target channel has no non-specific amplification, and the blank specificity of the kit is good.
Comparative example 1, remaining poorly performing primers and probes designed according to the invention
Because of the base-pairing rules, dimers are formed between the primer and/or probe, but with little probability, this can be eliminated at the beginning of the design. However, when multiple pathogens are jointly detected, a plurality of primers and probes are arranged, dimers are easy to occur between the primers and the primers, between the probes and the probes or between the primers and the probes, so that the conservation of design (which is crucial to the accuracy of detection) is ensured, and the mutual interference among different primer probes is considered, so that the primer probes need to be carefully designed.
Therefore, the inventors have also devised that the remaining primers and probes (sequences not shown) constitute different detection systems 1, 2 and 3, as well as for the detection of novel coronaviruses and monkey pox. The specific detection results are shown in fig. 6 to 8, and it can be seen from the figures that only part of amplification curves appear in the detection, the amplification curves have low amplification and poor repeatability, and other targets even have no amplification curves, so that the overall detection effect is poor.
Claims (10)
1. A novel composition of combined coronavirus and monkey poxvirus assays, said composition comprising simultaneously:
an upstream primer and a downstream primer and a probe for detecting 2019-nCoV ORF1ab genes as shown in SEQ ID NO. 1-3;
an upstream primer and a downstream primer and a probe for detecting 2019-nCoV N gene as shown in SEQ ID NO. 4-6; and
the upstream and downstream primers and probes for detecting the monkey poxvirus are shown as SEQ ID NO. 7-9.
2. The composition of claim 1, wherein the composition further comprises:
the upstream and downstream primers and probes for detecting the monkey poxvirus are shown as SEQ ID NO. 10-12.
3. The composition according to claim 1 or 2, wherein the upstream and downstream primers and probes for detecting an internal standard are an internal standard upstream primer shown in SEQ ID NO. 13, an internal standard downstream primer shown in SEQ ID NO. 14, and an internal standard probe shown in SEQ ID NO. 15.
4. A composition according to claim 3, wherein the fluorophores of the composition probes are different from each other and do not interfere with each other.
5. The composition of claim 4, wherein the fluorescent reporter group of the 2019-nCoV ORF1ab gene probe is FAM; the fluorescence reporter group of the 2019-nCoV N gene probe is HEX; the fluorescent reporter group of the monkey pox virus probe is ROX; the fluorescence reporter group of the internal standard probe is CY5.
6. The composition according to any one of claims 1-2, 4-5, wherein the components of the composition are present in a mixed form.
7. Use of a composition according to any one of claims 1 to 6 for the preparation of a kit for the joint detection of novel coronaviruses and monkey poxviruses.
8. A kit for the joint detection of novel coronaviruses and monkey pox viruses comprising a composition according to any one of claims 1 to 6.
9. The kit of claim 8, further comprising a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs, a reverse transcriptase, a uracil glycosylase, DNA polymerase, PCR buffer and Mg 2+ At least one of them.
10. A method for joint detection of novel coronaviruses and monkey pox for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing a fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 6 or the kit of any one of claims 8 to 9;
3) The results were obtained and analyzed.
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