CN116716435A - Composition for detecting novel coronavirus RNA - Google Patents

Composition for detecting novel coronavirus RNA Download PDF

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CN116716435A
CN116716435A CN202310232772.7A CN202310232772A CN116716435A CN 116716435 A CN116716435 A CN 116716435A CN 202310232772 A CN202310232772 A CN 202310232772A CN 116716435 A CN116716435 A CN 116716435A
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novel coronavirus
gene
solution
sample
composition
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王海滨
高智强
杨晓涵
彭闯
周其玲
窦瑞艳
王奇
唐洁
张公安
石风
王维
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Beijing Nagene Diagnostic Reagent Co ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/701—Specific hybridization probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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Abstract

The invention discloses a composition for detecting novel coronavirus RNA. The composition is used for detecting novel coronavirus RNA in a specimen, the minimum detection limit can reach 50copies/mL, and the Ct value standard is 38; the kit disclosed by the invention simultaneously comprises an endogenous internal standard detection system, a special sample preservation solution and magnetic beads, is used for monitoring a sample collection process, a nucleic acid extraction process and a PCR amplification process, can reduce the occurrence of false negative results, and has good practical application value.

Description

Composition for detecting novel coronavirus RNA
Technical Field
The invention relates to the field of molecular diagnosis, in particular to a composition for detecting novel coronavirus RNA.
Background
Common pathogen detection methods include pathogen culture, serum antibody detection, nucleic acid detection and the like, wherein virus culture detection takes days and has low efficiency; serum antibodies have lower detection specificity. Therefore, the most common detection means in clinic are nucleic acid detection methods with shorter time, stronger specificity and higher sensitivity. There are a number of methods for detecting SARS-CoV-2 nucleic acid, including polymerase chain reaction (polymerase chain reaction, PCR), loop-mediated isothermal amplification, CRISPR/Cas system, etc. Among them, the PCR technology is the earliest developed nucleic acid amplification technology, and targets can be amplified by tens of thousands to hundreds of millions of times through multiple cycles of high temperature denaturation, low temperature annealing, and primer extension. As a relatively mature nucleic acid amplification technique, PCR technology has been widely used in many fields of life sciences.
Nucleic acid detection is now the preferred detection technique for SARS-CoV-2 as an important method for clinical examination. Compared with the traditional virus separation detection, the nucleic acid detection has the advantages of short time consumption, simple operation, lower experimental conditions, good stability and the like; compared with the conventional antigen and antibody detection, the method has the advantages of high sensitivity and specificity, no window period, difficult interference and the like.
The TaqMan probe method is based on the principle that an oligonucleotide probe capable of being specifically combined with a target is added into a reaction system, two ends of the oligonucleotide probe are respectively connected with a fluorescent group and a quenching group, and fluorescence is not generated due to fluorescence energy resonance transfer phenomenon when the two are coexistent. During primer extension, a DNA polymerase having 5' to 3' exonuclease activity cleaves the fluorescent group at the 5' end of the probe, thereby generating fluorescence. The presence or absence of the target molecule can be determined based on the number of amplification cycles (Ct value).
The genetic material of SARS-CoV-2 is single-stranded RNA, and the nucleic acid detection process comprises two steps of reverse transcription and PCR amplification. Firstly, the viral RNA is reverse transcribed into cDNA by reverse transcriptase, and then the cDNA is amplified by DNA polymerase. And judging whether the sample contains SARS-CoV-2 nucleic acid or not according to the Ct value of the sample amplification curve. The recommended standard of the national institute of health and protection is that the Ct value is less than or equal to 37 and can be reported as positive; no Ct value or Ct is more than or equal to 40, and is negative; the experiment was repeated with Ct values between 37 and 40.
Disclosure of Invention
The technical problems to be solved are as follows:
the technical scheme solves the problems that the product sensitivity of detecting the novel coronavirus by utilizing the polymerase chain reaction in the prior art is low, false negative results are easy to occur, and the like.
The technical scheme provided by the invention is as follows:
a composition for detecting novel coronavirus RNA, comprising a primer pair for amplifying novel coronavirus ORF1ab gene by polymerase chain reaction and a primer pair for amplifying novel coronavirus N gene by polymerase chain reaction,
wherein, the primer pair for amplifying the novel coronavirus ORF1ab gene by polymerase chain reaction has the sequence as follows:
ORF1ab gene upstream primer sequence: 5'-cacttaaaaacacagtctgtacc-3' the number of the individual pieces of the plastic,
ORF1ab gene downstream primer sequence: 5'-gaacccatgcttcagtcagctga-3';
the primer pair for amplifying the novel coronavirus N gene by polymerase chain reaction has the sequence as follows:
n gene upstream primer sequence: 5'-gctagaatggctggcaat-3' the number of the individual pieces of the plastic,
n gene downstream primer sequence: 5'-gcttgagagcaaaatgtc-3'.
To better achieve the object of the present invention, in certain embodiments of the present invention, the composition further comprises a probe for detecting the novel coronavirus ORF1ab gene and/or the novel coronavirus N gene by polymerase chain reaction,
wherein, the sequence of the probe for detecting the ORF1ab gene of the novel coronavirus is as follows:
5'-gtggaaaggttatggctgtagttgtgatc-3'；
the sequence of the probe for detecting the novel coronavirus N gene is as follows:
5'-gatgctgctcttgctttgctgctgcttgac-3'。
in the present invention, any suitable fluorescent group and quenching group may be labeled on the probe. Preferably, in certain embodiments of the invention, the 5 'end of the probe of the ORF1ab gene is labeled with a FAM fluorophore and the 3' end is labeled with a TAMRA quencher; the 5 '-end of the probe of the N gene is marked with a VIC fluorescent group, and the 3' -end is marked with a TAMRA quenching group.
In another aspect of the invention there is provided the use of the above composition in the manufacture of a product for detecting or screening novel coronavirus RNAs in a sample, wherein the detection or screening is qualitative or quantitative.
In another aspect of the invention, there is provided a kit comprising the composition described above.
In the present invention, the kit further comprises conventional parts of a nucleic acid detection kit, such as buffers, enzymes, dntps, swabs, instructions, etc.
In order to reduce false negative results that may occur in the sample collection, nucleic acid extraction and PCR amplification processes, it is preferred that in certain embodiments of the invention the kit further comprises an internal standard rnase p system comprising a primer pair for amplifying the rnase p gene by polymerase chain reaction.
Wherein, the primer pair for amplifying the RNaseP gene by polymerase chain reaction has the sequence as follows:
primer sequence upstream of RNaseP gene: 5'-agatttggacctgcgagcg-3' the number of the individual pieces of the plastic,
primer sequence downstream of RNaseP gene: 5'-gagcggctgtctccacaagt-3'.
In order to better achieve the object of the present invention, in certain embodiments of the present invention, the internal standard RNaseP system further comprises a probe for detecting the RNaseP gene by polymerase chain reaction,
wherein, the sequence of the probe for detecting the RNaseP gene is as follows:
5'-ttctgacctgaaggctctgcgcg-3'。
preferably, in certain embodiments of the invention, the probe of the rnase p gene is labeled with a CY5 fluorophore at the 5 'end and a TAMRA quencher at the 3' end.
Preferably, in certain embodiments of the invention, the kit further comprises a nucleic acid sample holding fluid comprising a pseudovirus for protecting RNA, said pseudovirus having the sequence 5'-AGTGCCCCGGGAGGTCTCGTAGA-3'.
More preferably, the pseudovirus is placed in a betaine solution, wherein the concentration of betaine in the betaine solution is 0.01-0.1M, and the concentration of pseudovirus in the betaine solution is 10 ng/mL-40 ng/mL.
Preferably, in certain embodiments of the present invention, the nucleic acid sample preservation solution further includes magnetic beads for nucleic acid detection, and the method for preparing the magnetic beads comprises: the solution containing ferrous ions is reacted at proper temperature, and alkaline solution with pH value regulated and NH are added into the reaction solution 4 + Stopping the reaction until no more particles are precipitated, and drying the obtained particles to obtain the magnetic beads.
More preferably, the alkaline solution with the pH value adjusted and the solution containing NH are added dropwise 4 + The rate of mixing of the solutions of (a) is: dripping once every 10 minutes, wherein the dripping amount of each time is 1-5% of the total volume of reactants, and dripping for 22-30 times; wherein, the alkaline solution for regulating the pH value and the solution containing NH are added dropwise 4 + The mixed solution of the solution composition of (1) and (2) the concentration of the alkaline solution with the pH value adjusted is 1-2 mol/L, containsWith NH 4 + The concentration of the solution is 1-2 mol/L.
In particular, the kit of the invention may comprise:
nCoV reagent A, 96 human parts per cartridge, 160. Mu.L per tube X1; 1920 parts per box, 1.5mL per tube×2, main components: reverse transcriptase, taq DNA polymerase, dNTPs, primers, probes, and the like.
nCoV reagent B, 96 persons/box, 0.92 mL/tube X1; 1920 parts per box, 18.0mL per tube×1, main ingredients: RT-PCR Buffer, magnesium ion, etc.
3. Negative quality control, 96 human parts per box, 700 μl/tube×1; 1920 parts per box, 6.5mL per tube×1, main ingredients: sample dilutions.
4. The quality control of the male is 96 parts per box, and the filling amount is 700 mu L per tube multiplied by 1; 1920 parts per box, 6.5mL per tube×1, main ingredients: pseudoviruses containing ORF1ab, N and RNaseP genes.
5. Nucleic acid sample preservation solution.
The storage conditions and the validity period of the kit are as follows:
s1, preserving the kit at the temperature of-20+/-5 ℃ and keeping the effective period for 12 months.
S2, preserving the reagent at 2-8 ℃ for 2 weeks, and preserving the kit at 37 ℃ for 1 week; the kit is transported at the temperature of between 76 ℃ below zero and 30 ℃.
S3, opening the bottle of the reagent and repeatedly freezing and thawing for no more than 6 times.
The novel coronavirus nucleic acid detection kit trial instrument comprises: fluorescent quantitative PCR apparatus such as macro stone SLAN, spread spectrum X4, quantum 5, etc.
The novel coronavirus nucleic acid detection kit requires for a sample:
s1, applying sample types: a pharyngeal swab.
S2, sample collection: reference is made to "specimen collection" related provision for execution.
Pharyngeal swab: the plastic rod swab of the flocking fiber head is passed over the tongue root, the tonsils on two sides of a person to be collected are slightly rubbed back and forth for at least 3 times with force, then the swab head is rubbed up and down on the back wall of the pharynx for at least 3 times, the swab head is immersed into a tube containing virus preservation solution (isotonic saline solution, tissue culture solution or phosphate buffer solution can be used as well), the tail is discarded, and the tube cover is screwed. The pharyngeal swab may also be placed in the same tube as the nasopharyngeal swab.
S3, sample preservation: the collected sample can be stored for 24 hours at the temperature of 2-8 ℃, and the sample which needs to be stored for a long time is stored below-20 ℃.
S4, sample transportation: sample transport should comply with relevant biosafety regulations of the country with respect to the second class of pathogens.
The result judgment standard after the detection of the kit comprises the following conditions:
negative control: no CT values and no typical amplification curves;
positive control: CT value is less than or equal to 38, and a typical amplification curve appears.
The beneficial effects of the invention are as follows:
the composition is used for detecting novel coronavirus RNA in a specimen, the minimum detection limit can reach 50copies/mL, and the Ct value standard is 38; the kit disclosed by the invention simultaneously comprises an endogenous internal standard detection system, is used for monitoring a sample collection process, a nucleic acid extraction process and a PCR amplification process, can reduce false negative results, and has good practical application value. Meanwhile, the nucleic acid sample preservation solution containing the pseudoviruses in the invention does not use guanidine salt components to inactivate viruses, protect the nucleic acid from being damaged, and adsorb proteins and other impurities in the sample, so that purified nucleic acid is directly obtained for PCR detection, and the structural integrity of RNA can be maintained for more than 7 days under the conventional condition. The nano magnetic beads prepared by the method have the advantages of large particle size, good particle uniformity and dispersibility, strong magnetic response, strong magnetic bead size controllability and the like, can be used for various scenes with different requirements in biological separation, and particularly can obtain good effects in the purification of nucleic acid purification samples.
Detailed Description
The invention discloses a composition for detecting novel coronaviruses, and a person skilled in the art can properly improve the implementation of process parameters by referring to the content of the specification. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.
The following is a description of some of the terms appearing in the present invention.
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Example 1:
1. reagent preparation
Preparing PCR amplification reagent: prepared according to the proportion of 8.5 mu L of the nCoV reagent B to 1.5 mu L of the nCoV reagent A per person, and fully and uniformly mixed for use.
Component name Volume (mu L)
nCoV reagent A 1.5
nCoV reagent B 8.5
Wherein, reagent A is: reverse transcriptase, taq DNA polymerase, dNTPs, primers, probes.
ORF1ab gene upstream primer: 5'-cacttaaaaacacagtctgtacc-3' the number of the individual pieces of the plastic,
ORF1ab gene downstream primer: 5'-gaacccatgcttcagtcagctga-3';
n gene upstream primer: 5'-gctagaatggctggcaat-3' the number of the individual pieces of the plastic,
n gene downstream primer: 5'-gcttgagagcaaaatgtc-3'.
Probes for detecting the novel coronavirus ORF1ab gene:
5'-gtggaaaggttatggctgtagttgtgatc-3'；
probes for detecting novel coronavirus N genes:
5'-gatgctgctcttgctttgctgctgcttgac-3'。
reagent B is RT-PCR Buffer and magnesium ion.
The reagent C is as follows: nucleic acid sample preservation solution.
2. Sample processing and sample addition
And (3) storing the nucleic acid sample, the negative quality control product and the positive quality control product in the nucleic acid sample storage liquid.
2.1 nucleic acid extraction:
and taking 300 mu L of negative quality control product, positive quality control product and sample to synchronously extract nucleic acid. The procedure was followed according to the recommended kit instructions for nucleic acid extraction, with 60. Mu.L of eluate.
Negative quality control: sample diluent (deionized water)
Controlling the nature of yang: pseudoviruses containing ORF1ab, N and RNaseP genes; wherein, the liquid crystal display device comprises a liquid crystal display device,
primer sequence upstream of RNaseP gene: 5'-agatttggacctgcgagcg-3' the number of the individual pieces of the plastic,
primer sequence downstream of RNaseP gene: 5'-gagcggctgtctccacaagt-3'.
2.2 sample addition
And subpackaging 10 mu L of prepared PCR amplification reagent into each reaction hole, adding 20 mu L of purified sample nucleic acid, negative quality control product and positive quality control product respectively, sealing, reversing, fully mixing uniformly, and performing instantaneous centrifugation to perform PCR amplification.
Note that: the recommended PCR amplification tube cover is used for sealing, and the mixing is reversed, so that the amplification reagent and the nucleic acid to be detected are thoroughly mixed; if the PCR sealing film is used, the nucleic acid to be detected and the amplification reagent should be blown for 2-3 times to ensure thorough mixing of the nucleic acid to be detected and the amplification reagent, and the amplification is carried out after centrifugation.
3. Positive judgment value
According to the detection result of clinical samples, the ROC curve method is used for determining that the reference values of the ORF1ab gene and the N gene are Ct values less than or equal to 38.
4. Performance index of kit
4.1. The minimum detection limit of the kit is as follows: 50copies/mL.
4.2. Specificity: the kit is used for detecting the human coronavirus (HKU 1, OC43, NL63 and 229E), SARS coronavirus and MERS coronavirus; influenza a viruses H1N1, H3N2, H5N1, H7N9, influenza b Yamagata, victoria, respiratory syncytial virus A, B, parainfluenza viruses 1, 2, 3, rhinovirus A, B, C group, adenovirus 1, 2, 3, 4, 5, 7, 55, enterovirus A, B, C, D group, human metapneumovirus, EB virus, measles virus, human cytomegalovirus, rotavirus, norovirus, mumps virus, varicella-zoster virus; mycoplasma pneumoniae and chlamydia pneumoniae; legionella, pertussis, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae E7, streptococcus pyogenes, klebsiella pneumoniae, and Mycobacterium tuberculosis; aspergillus fumigatus, candida albicans, candida glabrata, cryptococcus neoformans and human genomic DNA did not cross-react.
4.3. Interference study: the interference samples at the critical positive level were validated, 20 μg/mL purified mucin, 90% blood, 100 μg/mL phenylephrine, 100 μg/mL oxymetazoline, 0.89% sodium chloride (with preservative), 50 μg/mL beclomethasone, 50 μg/mL dexamethasone, 100 μg/mL flunisolide, 100 μg/mL triamcinolone acetonide, 320 μg/mL budesonide, 100 μg/mL mometasone, 200 μg/mL fluticasone, 200 μg/mL histamine hydrochloride, 200U/mL interferon alpha, 100 μg/mL zanamivir, 100 μg/mL ribavirin, 100 μg/mL oseltamivir, 100 μg/mL peramivir, 200 μg/mL lopinavir, 50 μg/mL ritonavir, 100 μg/mL arbutin, 20 μg/mL levofloxacin, 100 μg/mL doxorubicin, 200 μg/mL amoxicillin, 200 μg/mL ceftriadimefon, 100 μg/mL sample of the interference kit.
4.4. Precision: detecting a medium/strong positive sample and a critical positive sample, wherein the positive detection rate is 100%, the Ct value variation coefficient (CV,%) is less than or equal to 5%, and the negative detection rate is 100%.
4.5 minimum detection limit:
the standard sample was diluted with a negative throat swab sample to prepare a throat swab median sample, a low value sample (minimum limit of detection sample). The kit is used for detection, and the low-value precision, the median precision and the lowest detection limit of a detection sample of the kit are researched to verify the detection performance of the kit. The results are shown in Table 1. The low value sample concentration of this group was diluted to 50copies/mL.
TABLE 1
4.6 enterprise reference validation: detecting negative reference products of enterprises, wherein the results are negative; detecting positive references of enterprises, wherein the results are positive; detecting a detection limit reference product of an enterprise, wherein the detection rate is more than or equal to 95%; and detecting the enterprise repeatability reference, wherein the Ct value variation coefficient (CV,%) is less than or equal to 5%.
The experimental result proves that the composition provided by the invention is used for detecting novel coronavirus RNA in a sample, and the minimum detection limit can reach 50copies/mL, and the Ct value standard is 38.
Example 2: verification of preservation Effect of nucleic acid sample preservation solution of the present invention
1. Instrument, reagent, sample:
the main instrument is as follows: real-time fluorescence quantitative PCR instrument produced by Shanghai Hongshi medical science and technology Co.
The main reagent comprises: new coronavirus SARS-CoV-2 nucleic acid detection kit (fluorescence PCR method) produced by Beijing Najie diagnostic reagent Co., ltd
Sample: incorporation of novel coronavirus (SARS-CoV-2) pseudovirus into throat swab solutions
2. Experimental protocol:
sample preservation solutions of three formulations were prepared according to the following protocol.
Component (A) Concentration range Formulation 1 Formulation 2 Formulation 3
Betaine (Anhydrous) 0.01-0.10M 0.01M 0.10M 0.02M
Sodium chloride or potassium chloride 0.05-0.5％ 0.05％ 0.5％ 0.1％
Tween-20 0.005％-0.05％ 0.005％ 0.05％ 0.01％
Pseudovirus 10ng/mL-30ng/mL 10ng/mL 30ng/mL 20ng/mL
Magnetic bead 1％-2％ 1％ 2％ 1.5％
Adding novel coronavirus (SARS-CoV-2) pseudovirus respectively, standing at normal temperature (2-30deg.C) for 1 day, 4 days, 7 days, and 10 days, taking out corresponding experimental groups, standing at-20deg.C, and synchronously performing PCR detection after all four groups are completed.
3. Experimental results: as shown below.
The experimental results show that the scheme 3 in the three formulas has the optimal effect, and the detection result is still stable after being placed for 10 days at normal temperature.
Example 3: the inactivation effect verification of the nucleic acid sample preservation solution
1. Instrument, reagent, sample:
the main instrument is as follows: real-time fluorescence quantitative PCR instrument produced by Shanghai Hongshi medical science and technology Co.
The main reagent comprises: new coronavirus SARS-CoV-2 nucleic acid detection kit (fluorescence PCR method) produced by Beijing Najie diagnostic reagent Co., ltd
Sample: incorporation of novel coronavirus (SARS-CoV-2) pseudovirus into throat swab solutions
2. Experimental protocol:
preparing a sample preservation solution of the respiratory RNA virus without the pseudovirus, wherein the preparation method comprises the following steps:
reagent(s) Formulation of
Betaine (Anhydrous) 0.02M
Sodium chloride or potassium chloride 0.1％
Tween-20 0.01％
Pseudoviruses of the invention —
Magnetic bead 1.5％
The prepared sample preservation solution (experimental group) without the pseudovirus of the invention is compared with the traditional Hanks preservation solution (control group) without the inactivation function, and 100 mu L of the sample preservation solution is respectively taken out for detection after the novel coronavirus (SARS-CoV-2) pseudovirus with the same concentration is added, and the stock solution and 1 hour, 2 hours and 3 hours are taken out.
3. Experimental results: as shown below.
The experimental result shows that the virus content in Hanks preservation solution has no obvious change, but the virus content in sample preservation solution without pseudovirus of the invention is obviously reduced along with the time increase, which indicates that the sample preservation solution has hypotonic degreasing effect, can physically destroy virus structure and release virus RNA, and is easy to be degraded and destroyed by RNase after the release of the virus RNA.
Example 4: RNA protection function verification of nucleic acid sample preservation solution
The pseudovirus in the invention can competitively consume the RNase in the sample, thereby avoiding the degradation of the viral RNA in the sample and protecting the RNA to be detected. The presence of the pseudoviruses of the present invention does not affect the inactivation of other components of the present invention to perform hypotonic degreasing.
1. Instrument, reagent, sample:
the main instrument is as follows: real-time fluorescence quantitative PCR instrument produced by Shanghai Hongshi medical science and technology Co.
The main reagent comprises: new coronavirus SARS-CoV-2 nucleic acid detection kit (fluorescence PCR method) produced by Beijing Najie diagnostic reagent Co., ltd
Sample: incorporation of novel coronavirus (SARS-CoV-2) pseudovirus into throat swab solutions
2. Experimental protocol:
control group: adding a novel coronavirus (SARS-CoV-2) pseudovirus into a sample preservation solution without the pseudovirus of the invention;
experimental group: the novel coronavirus (SARS-CoV-2) pseudovirus is added into the sample preservation solution
3. Experimental results: as shown below.
As can be seen from experimental results, according to the experimental scheme, the results show that if no RNA protective substance exists, nucleic acid in the collected sample is completely degraded after 3 hours, and the results in the sample using the sample preserving fluid have no obvious change, so that the sample preserving fluid has a reliable virus inactivating function through the competitive occupation principle.
Example 5: preparation of ferromagnetic magnetic beads in nucleic acid sample preservation solution of the present invention
100mL of water was weighed into a 500mL three-necked flask, and 3.80g of FeCl was added 2 ·4H 2 0, general person N 2 Ultrasonic dissolution was performed for 10min. After dissolution, the solution was sonicated for another 10min. Rapidly adding 9mL of ammonia water and 1mL of 1mol/L NH 4 SO 4 The solution was stirred vigorously, after which 1mol/L (NH) of aqueous ammonia solution was added every 10 minutes 4 ) 2 SO 4 The solution was added once, 1mL each time, 10mL in total, and after all additions were completed, the reaction was stirred under nitrogen for another 30min.
Fe is observed to be 3 O 4 After the particle formation, 5mL of oleic acid was slowly added (the addition was completed within 30 min), and l h was vigorously stirred at a temperature of 70 ℃. The resulting product was washed with water and acetone in sequence multiple times until the magnetic nanoparticles were seen. The obtained product is dried for standby.
And weighing 0.74g of dried magnetic fluid powder, dissolving the magnetic fluid powder in 6mL of petroleum ether, and carrying out ultrasonic treatment on the obtained product for about 10min to uniformly disperse the product. 550. Mu.L of undecylenic acid was added in 24mL of water using a pipette and the pH was adjusted to 8.0 using NaOH solution.
And (3) detecting magnetism of the magnetic beads, sucking out the magnetic bead suspension into a 1.5mL centrifuge tube, and detecting magnetism of the magnetic beads, wherein the prepared magnetic beads are completely adsorbed to one side within two seconds to be qualified.
According to JY/T010-1996 (general rule of analytical scanning electron microscope method), S-4800 type cold field emission scanning electron microscope is adopted to analyze the magnetic microsphere of the detected object, the sample is diluted by absolute ethyl alcohol and then dispersed by ultrasonic, and is dropped on the surface of a clean silicon wafer, and the sample is naturally dried and then placed into a sample chamber.
Experimental example 6: magnetic property determination of ferromagnetic magnetic beads in nucleic acid sample preservation solution of the invention
Taking the nano magnetic beads prepared in the example 1, and drying in vacuum to obtain magnetic Fe 3 O 4 The particles were magnetically measured for magnetic properties using a vibrating sample magnetometer (VSM 7300 Lake Shore Cryotronics, inc). The saturation magnetic intensity analysis was performed on the nano-magnetic beads prepared in example 1, commercially available nano-magnetic bead 1 (magnoMind magnetic microsphere), commercially available nano-magnetic bead 2 (zhichuan microsphere), commercially available nano-magnetic bead 3 (Beisi le magnetic microsphere), and commercially available nano-magnetic bead 4 (Yi Jia nano-microsphere) by using VSM, and as a result, the MS values of the nano-magnetic beads prepared in example 1, commercially available nano-magnetic bead 2, commercially available nano-magnetic bead 3, and commercially available nano-magnetic bead 4 were about 65.2, 57.6, 46.2, 51.5, and 44.8 A.m, respectively 2 /kg. The magnetic properties of the nano-magnetic beads prepared in example 1 were demonstrated to be stronger than those of commercially available nano-magnetic beads.
Experimental example 7: nucleic acid detection experiment of ferromagnetic magnetic beads in nucleic acid sample preservation solution
1. Reagent preparation
The nucleic acid detection kit used in this experimental example was a novel coronavirus nucleic acid detection kit (novel coronavirus (2019-nCoV) nucleic acid detection kit (fluorescence PCR method)) from beijing wig diagnostic reagent limited.
Preparing PCR amplification reagent: prepared according to the proportion of 8.5 mu L of the nCoV reagent B to 1.5 mu L of the nCoV reagent A per person, and fully and uniformly mixed for use.
2. Sample treatment and sample application Using the nanomagnetic beads prepared in example 1
1) Nucleic acid extraction:
and taking 300 mu L of negative quality control product, positive quality control product and sample to synchronously extract nucleic acid. The procedure was followed according to the recommended kit instructions for nucleic acid extraction, with 60. Mu.L of eluate.
2) Sample addition
And subpackaging 10 mu L of prepared PCR amplification reagent into each reaction hole, adding 20 mu L of purified sample nucleic acid, negative quality control product and positive quality control product respectively, sealing, reversing, fully mixing uniformly, and performing instantaneous centrifugation to perform PCR amplification. The concentration dilution gradient of the sample is shown below.
PCR amplification
1) And (3) setting a circulation parameter: SLAN, kunpeng Archimed X4 and LC480
QuantStudioTM5
2) The reaction system was 30. Mu.L, and the fluorescence channel detection was selected as follows:
channel A (ORF 1ab gene) B (internal standard) C (N gene)
Detection of fluorescence FAM HEX/VIC CY5
4. Baseline and threshold settings
The baseline is usually the baseline automatically set by the instrument. Baseline adjustment principle: the region with stable fluorescent signal before exponential amplification is selected, the starting point (Start) avoids signal fluctuation in the initial stage of fluorescent collection, and the End point (End) is reduced by 1-2 cycles compared with the Ct value of a sample with exponential amplification at the earliest occurrence. The threshold setting principle is that the threshold line just exceeds the highest point of the negative quality control detection fluorescence curve.
5. Positive judgment value
The detection results are shown below. According to the detection result of clinical samples, the ROC curve method is used for determining that the reference values of the ORF1ab gene and the N gene are Ct values less than or equal to 38.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A composition for detecting novel coronavirus RNA, characterized in that the composition comprises a primer pair for amplifying novel coronavirus ORF1ab gene by polymerase chain reaction and a primer pair for amplifying novel coronavirus N gene by polymerase chain reaction,
wherein, the primer pair for amplifying the novel coronavirus ORF1ab gene by polymerase chain reaction has the sequence as follows:
ORF1ab gene upstream primer sequence: 5'-cacttaaaaacacagtctgtacc-3' the number of the individual pieces of the plastic,
ORF1ab gene downstream primer sequence: 5'-gaacccatgcttcagtcagctga-3';
the primer pair for amplifying the novel coronavirus N gene by polymerase chain reaction has the sequence as follows:
n gene upstream primer sequence: 5'-gctagaatggctggcaat-3' the number of the individual pieces of the plastic,
n gene downstream primer sequence: 5'-gcttgagagcaaaatgtc-3'.
2. The composition of claim 1, wherein the composition further comprises a probe for detecting the novel coronavirus ORF1ab gene and/or the novel coronavirus N gene by polymerase chain reaction,
wherein, the sequence of the probe for detecting the ORF1ab gene of the novel coronavirus is as follows:
5'-gtggaaaggttatggctgtagttgtgatc-3'；
the sequence of the probe for detecting the novel coronavirus N gene is as follows:
5'-gatgctgctcttgctttgctgctgcttgac-3'。
3. the composition of claim 2, wherein the probe of the ORF1ab gene is labeled with FAM fluorophore at the 5 'end and TAMRA quencher at the 3' end; the 5 '-end of the probe of the N gene is marked with a VIC fluorescent group, and the 3' -end is marked with a TAMRA quenching group.
4. Use of a composition as claimed in any one of claims 1 to 3 in the manufacture of a product for detecting or screening novel coronavirus RNAs in a sample, wherein the detection or screening is qualitative or quantitative.
5. Use of a composition as claimed in any one of claims 1 to 3 for the preparation of a product for diagnosing the presence or absence of a novel coronavirus infection in a patient.
6. A kit comprising a composition as claimed in any one of claims 1 to 3.
7. The kit of claim 6, further comprising a nucleic acid sample holding fluid comprising a pseudovirus for protecting RNA, said pseudovirus having a sequence of 5'-AGTGCCCCGGGAGGTCTCGTAGA-3';
preferably, the pseudovirus is placed in a betaine solution, wherein the concentration of betaine in the betaine solution is 0.01-0.1M, and the concentration of pseudovirus in the betaine solution is 10 ng/mL-40 ng/mL.
8. The kit according to claim 7, wherein the nucleic acid sample preservation solution further comprises magnetic beads for nucleic acid detection, and the preparation method of the magnetic beads is as follows: the solution containing ferrous ions is reacted at proper temperature, and alkaline solution with pH value regulated and NH are added into the reaction solution 4 + Stopping the reaction until no more particles are precipitated, and drying the obtained particles to obtain the magnetic beads;
preferably, the alkaline solution with the pH value adjusted and the solution containing NH are added dropwise 4 + The rate of mixing of the solutions of (a) is: dripping once every 10 minutes, wherein the dripping amount of each time is 1-5% of the total volume of reactants, and dripping for 22-30 times; wherein, the alkaline solution for regulating the pH value and the solution containing NH are added dropwise 4 + In the mixed solution composed of the solutions, the concentration of the alkaline solution with the pH value adjusted is 1-2 mol/L, and NH is contained 4 + Concentration of the solution of (2)1 to 2mol/L.
9. Use of a kit as claimed in any one of claims 6 to 8 in the manufacture of a product for detecting or screening for a novel coronavirus in a sample, wherein the detection or screening is qualitative or quantitative.
10. Use of a kit as claimed in any one of claims 6 to 8 for the preparation of a product for diagnosing the presence or absence of a novel coronavirus infection in a patient.
CN202310232772.7A 2023-03-07 2023-03-07 Composition for detecting novel coronavirus RNA Pending CN116716435A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117344062A (en) * 2023-12-06 2024-01-05 江苏硕世生物科技股份有限公司 Kit for rapidly detecting novel coronavirus nucleic acid and application method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117344062A (en) * 2023-12-06 2024-01-05 江苏硕世生物科技股份有限公司 Kit for rapidly detecting novel coronavirus nucleic acid and application method
CN117344062B (en) * 2023-12-06 2024-03-22 江苏硕世生物科技股份有限公司 Kit for rapidly detecting novel coronavirus nucleic acid and application method

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