CN114410836A - Kit and method for detecting human parvovirus B19 by integrating sample collection treatment, nucleic acid extraction and multiple isothermal amplification - Google Patents

Kit and method for detecting human parvovirus B19 by integrating sample collection treatment, nucleic acid extraction and multiple isothermal amplification Download PDF

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CN114410836A
CN114410836A CN202111554876.7A CN202111554876A CN114410836A CN 114410836 A CN114410836 A CN 114410836A CN 202111554876 A CN202111554876 A CN 202111554876A CN 114410836 A CN114410836 A CN 114410836A
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CN114410836B (en
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李敏
汪骅
王亚楠
黄芊
马硝惟
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Renji Hospital Shanghai Jiaotong University School of Medicine
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Abstract

The invention provides a kit and a method for detecting human parvovirus B19, which integrate sample collection treatment, nucleic acid extraction and multiple isothermal amplification. The kit comprises a constant-temperature amplification detection reagent containing a primer with a nucleotide sequence of SEQ ID NO. 3-10, a reagent for nucleic acid separation/purification, a negative control and a positive control; the isothermal amplification detection reagent and the reagent for nucleic acid separation/purification are converted into lyophilized powder by a special lyophilization process. The method provided by the invention rapidly obtains the DNA of the sample to be detected through the PCR reaction tube containing the micro separation tube, and accurately detects the human parvovirus B19 by adopting a multiple constant temperature probe method. The kit and the method provided by the invention overcome one or more defects of the existing constant temperature amplification method, and particularly realize 'sample in and result out' in a real sense; the detection sensitivity is obviously improved while the storage and transportation cost is reduced, the POCT detection is convenient to realize, and the POCT detection kit can be widely applied to hospital emergency treatment and basic medical institutions.

Description

Kit and method for detecting human parvovirus B19 by integrating sample collection treatment, nucleic acid extraction and multiple isothermal amplification
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit and a method for detecting human parvovirus B19, which integrate sample collection processing, nucleic acid extraction and multiple isothermal amplification.
Background
The human parvovirus B19 is the smallest known virus, belongs to parvoviridae, is a single-stranded DNA virus consisting of no envelope and 3500 bases, belongs to the parvoviridae, is one of erythroviruses, has the diameter of 20-25 nm and has very strong resistance to external physical and chemical factors. The B19 virus can be divided into three genotypes of 1, 2 and 3, wherein the 1 type and the 3 type are respectively divided into two subtypes.
B19 viral infections are ubiquitous and can be transmitted by droplets, skin contact, blood products or vertically from the placenta. The clinical manifestations of the disease vary among people of different ages and immune states, and can cause infectious erythema, fetal edema and even death, acute arthritis, chronic anemia, aplastic crisis and other diseases. In addition, researchers have found that some cases of kidney disease, hepatitis, neurological diseases, myocardial damage and some autoimmune diseases are also associated with infection by the B19 virus.
Common methods for detecting human parvovirus B19 infection rely on antigen, antibody and nucleic acid detection. The detection of the B19 virus antibody has a window period waiting for the generation of the antibody for 2-4 weeks, the antigen detection sensitivity is low, the nucleic acid detection is directed at the DNA of the B19 virus, the detection time is not limited, the sensitivity is highest, the presence of a trace amount of B19 virus nucleic acid in a sample can be detected, and the method has great advantages in the detection method of the B19 virus. The B19 nucleic acid detection method mainly comprises a PCR detection technology and a constant temperature detection technology, however, the PCR detection technology has the following problems: (1) PCR detection generally depends on repeated operations of multiple stages such as denaturation, annealing, extension and the like, generally takes longer time, and needs 1.5-2 hours for one-time detection; (2) POCT on-site detection is difficult to realize, so that the PCR detection technology is not popularized and applied in the basic level.
At present, no products for detecting human parvovirus B19 on the market exist, and the isothermal amplification technology is used for completing the amplification reaction of nucleic acid at a constant temperature, so that only a simple isothermal device is needed, the repeated operation of multiple stages such as denaturation, annealing and extension is not needed, the amplification efficiency can be improved, and the detection time can be shortened. Isothermal amplification techniques can be classified into nucleic acid sequence-dependent amplification (NASBA), loop-mediated isothermal amplification (LAMP), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), and Recombinase Polymerase Amplification (RPA) according to their technical principles. The NASBA technology is more suitable for isothermal amplification of RNA as a template, and a denaturation process is required to be added for the amplification of DNA; the LAMP technology is easily limited by a sequence to be detected, and a proper primer combination cannot be designed, so that the method is limited and cannot be used; RCA techniques rely on circular templates, but the vast majority of genomic DNA is linear molecules; the Strand Displacement Amplification (SDA) technology requires a plurality of complicated stages such as preparation of a DNA single-strand template; HDA technology is limited by DNA helicases; RPA techniques tend to be harsh in reaction conditions and cannot amplify crude samples. The method usually adopts different result reading accompanying methods such as SYBR GREEN staining method, calcein method, turbidity method and the like, but the reading accompanying methods have congenital defects, and all use nucleic acid amplification products or byproducts as targets to directly detect, and positive results are also presented when nonspecific amplification occurs, so that false positive is easily caused, and multiple detection is difficult to realize. The detection of the human parvovirus B19 usually needs to introduce an internal standard to carry out quality control on the sample collection and detection process so as to avoid false negative, the detection system is a multiple detection system, one fluorescence channel corresponds to the human parvovirus B19 nucleic acid, and the other fluorescence channel corresponds to the internal standard. Recently, enterprises introduce Taq-Man probes with neck ring structures to realize multiple fluorescence detection of isothermal PCR, but the Taq-Man probes with neck ring structures are difficult to design.
The target gene of isothermal amplification is a nucleic acid fragment, the nucleic acid of B19 virus is wrapped in a protein capsid, and the virus exists in a clinical sample with complex components, so the clinical sample needs to be processed before amplification, and the nucleic acid can be amplified after being released and purified. At the present stage, nucleic acid of a sample is extracted mainly by a centrifugal column method and a magnetic bead method, the two methods are subjected to steps of cracking, combining, washing, rinsing, eluting and the like, the operation is complex, the time consumption is long, and a large amount of protein allosteric effectors and organic solvents are required. The sample extraction easily causes cross contamination among samples to cause false positive, and meanwhile, the nucleic acid extraction also needs professional laboratory equipment and environment and has special requirements on operators.
The constant temperature amplification system contains active ingredients such as polymerase, dNTP and the like, needs to be stored at low temperature, needs to be unfrozen and repeatedly frozen and thawed when in use, is complex to operate, and can generate certain influence on active ingredients in a reagent and even inactivate the active ingredients. In addition, cryopreservation of diagnostic reagents can increase costs in storage, transportation, and energy consumption. Freeze drying is a drying method in which a material containing water to be dried is frozen into a solid, and the material is dehydrated at a low temperature by utilizing the sublimation property of water under the condition of low temperature and reduced pressure to achieve the purpose of drying. The freeze drying technology has the following advantages: 1) many heat-sensitive substances do not denature or deactivate; 2) during the freeze-drying process, the growth of microorganisms and the action of enzymes cannot be carried out, so that the original properties can be maintained; 3) the volume is almost unchanged, the original structure is kept, and the concentration phenomenon cannot occur; 4) the drying is carried out under vacuum, so that little oxygen is generated, and some substances which are easy to oxidize are protected; 5) can eliminate over 95-99% of water and can make the dried product be stored for a long period without deterioration.
Therefore, there is an urgent need to develop a rapid-extraction multiple isothermal probe method for detecting human parvovirus B19, which can perform reagent lyophilization, to overcome one or more of the disadvantages of the existing isothermal amplification methods, achieve multiple isothermal amplification detection of "sample in and out", and achieve normal temperature storage and transportation of detection reagents.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a kit and a method for detecting human parvovirus B19, which integrate sample processing, nucleic acid extraction and multiple isothermal amplification.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer composition for detecting human parvovirus B19 at normal temperature, which comprises a primer aiming at the nucleotide sequence of human parvovirus B19 and having a nucleotide sequence of SEQ ID NO. 3-6 and a primer aiming at the nucleotide sequence of an internal standard beta globin gene (hereinafter referred to as HBB) and having a nucleotide sequence of SEQ ID NO. 7-10, wherein N in the sequences is 3-methyladenine.
Furthermore, primers with nucleotide sequences of SEQ ID NO. 3 and SEQ ID NO. 7 are specific fluorescent probes, tetrahydrofuran modification sites are introduced into the fluorescent probes, and a fluorescent reporter group and a fluorescent quenching group are coupled in the vicinity of the tetrahydrofuran modification sites.
Further, the primers with the nucleotide sequences of SEQ ID NO. 3 and SEQ ID NO. 7 are respectively:
SEQ ID NO:3：CCNCTNTGNNNNCTGGGCNATAAACTACACTTTTGATTTCCCT(FAM-dT)(THF)(BHQ1-dT)GGAATTAATGC-PO4；
SEQ ID NO:7：TGNNGGCTCNTGGCNAGNAAGTGCTCGGTGCCTTTAGT(JOE-dT)(THF)(BHQ1-dT)GATGGCCTGGC-PO4。
in a second aspect, the invention provides a kit for detecting human parvovirus B19, which integrates sample collection treatment, nucleic acid extraction and multiple isothermal amplification, and comprises an isothermal amplification detection reagent containing the primer composition provided by the first aspect of the invention, a reagent for nucleic acid isolation/purification, a negative control and a positive control;
the constant temperature amplification detection reagent also comprises dNTP and Mg2+Buffer, Bsu DNA polymerase, methylpurine DNA glycosylase (AAG) and Endonuclease IV enzyme (derived from bacteria)Endonuclease, and more preferentially double-stranded DNA at the 3' -concave end, involved in DNA damage repair, hereinafter referred to as Nfo enzyme);
the above-mentioned reagent for nucleic acid separation/purification comprises chelex100 chelate resin, NaOH, sodium dodecyl sulfate and an internal standard HBB linearized plasmid.
Further, the isothermal amplification detection reagent comprises the following components in concentration: 20mM-50mM NaOH, 0.3-0.6% PEG8000, 4-5mM magnesium chloride, 10-30mM Tris-HCl (pH 8.2), 30-50mM KCl, 10-20mM ammonium sulfate, 200. mu.M-400. mu.M dATP, 200. mu.M-400. mu.M dGTP, 200. mu.M-400. mu.M dCTP, 100. mu.M-200. mu.M dTTP, 100. mu.M-200. mu.M dUTP, 0.1-0.3. mu.M of each primer, 0.005-0.01U/. mu.L Nfo enzyme, 0.05-0.1U/. mu.L methylpurine DNA glycosylase (AAG), and 0.3-0.5U/. mu.L DNA polymerase.
Further, the reagent for nucleic acid isolation/purification includes the following components at final concentrations: 2 to 5 percent of chelex100 chelating resin, 0.2 to 0.5 percent of NaOH, 0.1 to 0.5 percent of sodium dodecyl sulfate and 10 percent of sodium dodecyl sulfate2-103Copies/. mu.L of internal standard HBB linearized plasmid.
Further, the constant temperature amplification detection reagent and the reagent for nucleic acid separation/purification are in a freeze-dried powder shape; the isothermal amplification detection reagent also comprises a freeze-drying protective agent, which comprises: 1% -5% glycogen (glycogen), 1% -5% bovine serum albumin and 0.1-0.2 ng/. mu.L carrier RNA (carrier RNA).
Further, the preparation method of the freeze-dried powder isothermal amplification detection reagent comprises the following steps:
(1) preparation of nucleic acid amplification reagents: adding NaOH, PEG8000, magnesium chloride, Tris-HCl (pH 8.2), KCl, ammonium sulfate, calf serum albumin, dATP, dGTP, dCTP, dTTP, dUTP, primers, AAG, Bsu DNA polymerase, Nfo enzyme and sterilized purified water into a centrifuge tube according to a certain proportion, and uniformly mixing;
(2) preparing a freeze-drying protective agent: weighing a certain amount of glycogen and bovine serum albumin, adding the glycogen and the bovine serum albumin into the sterilized purified water for dissolving, adding carrier RNA, and fixing the volume to the final volume;
(3) taking out each constant-temperature amplification reagent and the freeze-drying protective agent according to the volume of 100: (3-5), and subpackaging the mixture into PCR reaction tubes according to the reaction amount of 40 mu L;
(4) and (3) immersing the PCR reaction tube into liquid nitrogen for pre-freezing for 2-5 minutes, and directly putting the PCR reaction tube into a freeze dryer for vacuum freeze drying.
Further, the negative control is sterilized normal saline; the positive control was 103copies/uL of pseudovirus containing the B19 sequence.
In a third aspect, the present invention provides a method for detecting human parvovirus B19 by integrating collection sample treatment, nucleic acid extraction and multiple isothermal amplification using the kit provided in the second aspect of the present invention, comprising the following steps:
the method comprises the following steps: releasing single-stranded DNA from a sample to be tested into a reaction tube containing a nucleic acid amplification reagent by centrifugation using the reagent for nucleic acid isolation/purification;
placing the reaction tube into a sample groove of an amplification instrument, setting positive control and negative control, and then carrying out amplification reaction;
and step three, automatically storing results after the reaction is finished, and analyzing the amplification curves of the detection target and the reference gene respectively.
Further, the centrifugation conditions in step one were 5000 rpm and 10 seconds.
Further, the fluorescence detection channel adopted in the second step is FAM/JOE; the condition of the amplification reaction is 10-40 ℃; 1 minute/cycle, 40 cycles; preferably, the amplification temperature is 37 ℃.
Further, the analysis process in the third step is as follows:
a. quality control: negative control: the FAM channels have no Ct value or Ct is more than 38; positive control: ct of both FAM and internal standard channel is less than or equal to 38; the above requirements are all required to be met in the same detection, otherwise, the detection is invalid and needs to be carried out again; and
b. and (5) judging a result:
firstly, whether the internal standard has a typical S-shaped amplification curve or not is analyzed, and Ct is less than or equal to 38, if yes, the detection is effective, and the subsequent analysis can be continued:
(1) if the typical S-type amplification curve is detected by the FAM channel and Ct is less than or equal to 38, the human parvovirus B19 is positive; if the FAM channel does not detect a typical S-type amplification curve, the human parvovirus B19 is negative;
(2) for positive samples, the internal standard detection result does not make requirements; and for a negative sample, detecting the internal standard of the sample to be positive, and if detecting the internal standard of the sample to be negative, detecting the detection result of the sample to be invalid.
In a fourth aspect, the present invention provides an apparatus for use in the method provided in the third aspect of the present invention, which is a PCR reaction tube containing a micro separation tube, comprising a separation tube containing a reagent for nucleic acid separation/purification at an upper portion and a PCR reaction tube containing a reagent for isothermal amplification at a lower portion; the separation tube is inserted into the lower PCR reaction tube to realize the connection of the two;
the bottom of the separation tube was a 0.45 μm filter.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention adopts a freeze-dried multiple constant temperature probe method detection reagent to detect the human parvovirus B19 nucleic acid, can overcome one or more defects of the existing constant temperature amplification method, and particularly realizes sample input and result output in a real sense. Compared with the existing human parvovirus B19 nucleic acid detection kit, the kit has the following advantages:
(1) the kit and the method provided by the invention realize the integration of nucleic acid extraction and detection, have the technical characteristics of 'detection in time, sample in and result out', are convenient for realizing POCT detection, and can be widely applied to hospital emergency treatment and basic medical institutions;
(2) the invention adopts the constant temperature reaction, avoids the dependence of PCR technology on temperature precision control equipment, and reduces the cost of experimental instruments; the three enzymes can be detected at normal temperature, so that the dependence on heating equipment is eliminated; the primer is simple in design, only 2 regions of a template to be detected need to be covered, and the condition that constant-temperature amplification cannot be implemented due to the fact that a proper primer pair cannot be obtained in LAMP cannot occur;
(3) the invention adopts the freeze-drying protective agent with a special formula and the reagent freeze-drying process, not only can realize the stability of normal-temperature preservation activity, get rid of cold chain and reduce the storage and transportation cost, but also can ensure that a constant-temperature amplification system can amplify a crude sample and ensure that the freeze-dried powder can be quickly rehydrated and activated to realize second-time recovery and preserve the optimal activity, and meanwhile, after the reagent is freeze-dried, the volume of the added sample can be improved, and the detection sensitivity of the in-vitro diagnostic reagent can be obviously improved to 1 copy/reaction.
Drawings
FIG. 1 shows a schematic representation of Nfo enzymes cleaving a tetrahydrofuran modification site in a primer and releasing a fluorophore in one embodiment of the invention;
FIG. 2 is a schematic diagram showing the structure of an apparatus for sample processing and nucleic acid extraction according to an embodiment of the present invention; wherein, 1-separation tube, 2-PCR reaction tube, 3-reagent for nucleic acid separation/purification, 4-freeze-dried powder, 5-filter membrane;
FIG. 3 is a schematic diagram of the multi-isothermal probe method according to an embodiment of the present invention;
FIG. 4 shows amplification curves for detecting a target and a reference gene in one embodiment of the present invention; wherein panel a shows the amplification plot for B19 positive samples; FIG. B shows the amplification profile of a B19 negative sample with Cycle number (1 Cycle at 1 minute) on the abscissa and fluorescence on the ordinate;
FIG. 5 is a graph showing the amplification sensitivity of B19 nucleic acid detected by the multiplex isothermal probe method in one embodiment of the present invention, wherein the abscissa represents the number of cycles (1 Cycle for 1 minute) and the ordinate represents the fluorescence value.
Detailed Description
The invention provides a kit and a method for detecting human parvovirus B19, which integrate sample collection treatment, nucleic acid extraction and multiple isothermal amplification, and aims to provide a simple, efficient, high-specificity and high-sensitivity multiple isothermal amplification method for human parvovirus B19; in addition, the invention converts the detection system of the human parvovirus B19 from liquid state to solid state, so that the constant temperature amplification system realizes stable activity at normal temperature, gets rid of cold chain, reduces storage and transportation cost, and simultaneously, the volume of the added sample can be increased after the reagent is freeze-dried, thereby obviously improving the detection sensitivity of the in vitro diagnostic reagent.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
This example provides a kit for detecting human parvovirus B19, which integrates sample processing, nucleic acid extraction and multiple isothermal amplification, and comprises isothermal amplification detection reagents, reagents for nucleic acid isolation/purification, a negative control and a positive control. Wherein:
(1) isothermal amplification assay reagents included 50mM NaOH, 0.5% PEG8000, 4mM magnesium chloride, 20mM Tris-HCl (pH 8.2), 35mM KCl, 15mM ammonium sulfate, 200. mu.M dATP, 200. mu.M dGTP, 200. mu.M dCTP, 200. mu.M dTTP, 200. mu.M dUTP, 0.2. mu.M primer 1-primer 8, 0.1U/. mu.L methylpurine DNA glycosylase (AAG), 0.4U/. mu.L Bsu DNA polymerase, 0.008U/. mu.L Nfo enzyme.
The primers comprise a primer aiming at the target gene sequence of the human parvovirus B19 nucleic acid as SEQ ID NO. 1 and a primer aiming at the internal standard HBB gene as SEQ ID NO. 2 (shown in the following tables 1 and 2).
TABLE 1 human parvovirus B19 nucleic acid amplification primer sequences
Figure BDA0003418275710000071
Figure BDA0003418275710000081
Note: "N" is 3-methyladenine.
TABLE 2 internal standard HBB amplification primer sequences
Figure BDA0003418275710000082
Note: "N" is 3-methyladenine.
Wherein primer 1 comprises a primer complementary to the upstream end of the human parvovirus B19 DNA to be amplified, primer 2 comprises a primer complementary to the downstream segment of the human parvovirus B19 DNA to be amplified, primer 5 comprises a primer complementary to the upstream end of the internal standard HBB gene fragment to be amplified, primer 6 comprises a primer complementary to the downstream segment of the internal standard HBB gene fragment to be amplified, and each of primer 1, primer 2, primer 5 and primer 6 comprises a 3-methyladenine modified portion and an unmodified portion from the 5 'end to the 3' end, wherein at least the conventional base adjacent to the unmodified portion in the modified portion is replaced with a modified base; the sequence of primer 3 is identical to the sequence of the modified portion in primer 1, the sequence of primer 4 is identical to the sequence of the modified portion in primer 2, and the sequence of primer 7 is identical to the sequence of the modified portion in primer 5; the sequence of primer 8 is identical to the sequence of the modified portion in primer 6. After the 3-methyladenine modified base modified part is specifically recognized and cut by the enzyme which recognizes and cuts the 3-methyladenine modified base in one strand of the double-stranded DNA, the hybridization stability of the 3-methyladenine modified part and the DNA to be amplified can be effectively reduced.
In addition, the primer 1 and the primer 5 are specific fluorescent probes, the probes are oligonucleotides, 3-methyladenine modified base is introduced into a 5' segment, and the probe is used for isothermal amplification; a tetrahydrofuran modification site is introduced into the middle part, and a fluorescence reporter group and a fluorescence quenching group are coupled in the vicinity of the tetrahydrofuran modification site and are used for fluorescence detection. Specifically, the sequences of primer 1 and primer 5 are as follows:
primer 1: CCNCTNTGNNNNCTGGGCNATAAACTACACTTTTGATTTCCCT (FAM-dT) (THF) (BHQ1-dT) GGAATTAATGC-PO4；
Primer 5: TGNNGGCTCNTGGCNAGNAAGTGCTCGGTGCCTTTAGT (JOE-dT) (THF) (BHQ1-dT) GATGGCCTGGC-PO4。
As shown in FIG. 1, when primer 1 and primer 5 are intact, the fluorescent signal emitted from the reporter is absorbed by the quencher. When the probe and a target sequence structure form double-stranded DNA, Nfo enzyme meets tetrahydrofuran modification sites in the patrol process, the damage of the DNA is considered to be repaired, so the sites on the probe are sheared, fluorescent groups are released, the sheared probe has free 3' -OH, and the probe can be used as a primer to continuously synthesize a new template, and different fluorescence-labeled probes are used, so that multiple PCR detection can be realized.
(2) The reagent for nucleic acid isolation/purification includes the following components at final concentrations: 5% chelex100 chelating resin, 0.2% NaOH, 0.1% sodium lauryl sulfate and 10%2Copies/. mu.L of internal standard HBB linearized plasmid. Wherein, the sodium dodecyl sulfate and NaOH can denature the capsid of the B19 virus protein and release nucleic acid; chelex100 chelating resin is a chemical chelating resin composed of styrene and divinylbenzene copolymer, contains paired iminodiacetate ions, can chelate multivalent ions, has high affinity and chelation effect on high-valence metal ions in particular, and can adsorb impurities in serum/plasma samples.
(3) The negative control was sterile saline.
(4) The positive control was 103copies/uL of pseudovirus containing the B19 sequence.
Example 2
This example provides a kit for detecting human parvovirus B19 nucleic acid based on lyophilized multiple isothermal technology reagents based on example 1, wherein the isothermal amplification detection reagents and the reagents for nucleic acid isolation/purification are lyophilized powders as compared to example 1.
The isothermal amplification detection reagent also comprises a freeze-drying protective agent, and the freeze-drying protective agent comprises the following components: 2.5% glycogen, 2.5% bovine serum albumin and 0.1 ng/. mu.L carrier RNA. Wherein: the bovine serum albumin can effectively protect the activities of methylpurine DNA glycosylase (AAG), Bsu DNA polymerase and Nfo enzyme, and simultaneously, an amplification reaction system can be used for the amplification of a sample; the carrier RNA can effectively prevent the final concentration from being reduced due to the adsorption of the primers 1-8 on the tube wall, and the influence on constant-temperature amplification is avoided; glycogen can accelerate the redissolution speed of the freeze-dried powder.
Based on the above, the steps of preparing the freeze-dried isothermal amplification detection reagent and the reagent for nucleic acid separation/purification as freeze-dried powder are as follows:
(ii) a reagent preparation step for nucleic acid isolation/purification: adding chelex100 chelating resin, NaOH, sodium dodecyl sulfate, internal standard HBB linearized plasmid and sterilized purified water into a centrifugal tube according to a certain proportion, uniformly mixing, and storing at normal temperature for later use.
Preparing a nucleic acid amplification reagent: adding NaOH, PEG8000, magnesium chloride, Tris-HCl (pH 8.2), KCl, ammonium sulfate, calf serum albumin, dATP, dGTP, dCTP, dTTP, dUTP, primer 1-primer 8, AAG, Bsu DNA polymerase, Nfo enzyme and sterilized purified water into a centrifuge tube according to a certain proportion, mixing uniformly, and storing at-20 +/-5 ℃ for later use.
Preparing a freeze-drying protective agent: weighing a certain amount of glycogen and bovine serum albumin, adding the glycogen and the bovine serum albumin into the sterilized purified water for dissolving, adding carrier RNA, and fixing the volume to the final volume.
Preparing a liquid reagent for freeze-drying:
the reagents for nucleic acid isolation/purification were dispensed directly into PCR reaction tubes in an amount of 50. mu.L/reaction, and lyophilized.
The prepared human parvovirus B19 nucleic acid detection constant-temperature amplification reagent and the freeze-drying protective agent are mixed according to the volume of 100: 5, and subpackaging the mixture into PCR reaction tubes according to the amount of 40 mu L/reaction for freeze-drying treatment.
The freeze drying treatment comprises the following steps:
and (3) immersing the PCR reaction tube containing the freeze-dried liquid reagent into liquid nitrogen for prefreezing for 2-5min (note that the liquid nitrogen cannot enter the tube during prefreezing to ensure the cleanliness of the reagent), and directly putting the tube into a freeze dryer for vacuum freeze drying.
In particular, this example provides a kit (96 persons) having the composition shown in table 3 below.
TABLE 3 human parvovirus B19 nucleic acid assay kit Components
Figure BDA0003418275710000101
Figure BDA0003418275710000111
Example 3
Referring to FIG. 2, this example provides an apparatus for separating and purifying nucleic acid, which is a PCR reaction tube containing a micro separation tube, comprising a separation tube 1 located at the upper part and a PCR reaction tube 2 located at the lower part; the separation tube 1 is inserted into the lower PCR reaction tube 2 to realize the connection of the two; the bottom of the separation tube 1 is a 0.45 μm filter membrane 5.
A reagent 3 for nucleic acid separation/purification, specifically a lyophilized product for nucleic acid separation/purification containing chelex100 chelate resin, NaOH, sodium dodecyl sulfate and an internal standard HBB linearized plasmid as provided in example 2 above, is contained in the separation tube 1; the lyophilized powder 4 containing isothermal amplification reagent for detecting nucleic acid of human parvovirus B19, specifically the one containing dNTP, primer probe and Mg provided in the above example 2, is filled in the PCR reaction tube2+And reagents such as buffer solution, Bsu DNA polymerase, methylpurine DNA glycosylase (AAG), and Nfo enzyme.
The nucleic acid separation/purification reagent is formed into freeze-dried microspheres, and then the whole is transferred into a separation tube of a nucleic acid separation/purification device. And (3) placing the separation tube containing the freeze-dried microspheres on a PCR reaction tube containing the freeze-dried microspheres of the constant-temperature amplification reagent, and tightly covering the separation tube.
Example 4
The present embodiment provides a method for detecting human parvovirus B19 at room temperature using the kit provided in embodiment 2 and the device provided in embodiment 3, comprising the steps of:
(1) isolation and purification of nucleic acids
Sucking 50 mu L of a serum/plasma sample to be detected, adding the serum/plasma sample into a separation tube 1 containing a freeze-drying reagent, fully and uniformly mixing and dissolving freeze-drying microspheres in the separation tube, standing for 1min, rotating at 5000 rpm, centrifuging for 10 seconds, wherein chelex100 chelate resin adsorbing impurities cannot pass through a filter membrane 5 but is remained in the separation tube 1 on the upper layer; b19 virus nucleic acid without impurities is denatured into single-stranded DNA under alkaline condition at normal temperature, passes through the filter membrane 5, is released into the PCR reaction tube 2, and is fully mixed with freeze-dried microspheres dissolved in the PCR reaction tube 2, so that the separation and purification of nucleic acid are realized.
(2) Isothermal amplification
And placing the PCR reaction tube 2 into a sample groove of an amplification instrument, setting a positive control, a negative control and a sample to be detected in a corresponding sequence, and setting the name of the sample. In the PCR reaction tube 2, under the action of methyl purine DNA glycosylase (AAG) for identifying and excising 5' -end 3-methyl adenine in one strand of double-stranded DNA and Bsu DNA polymerase with strand displacement function, primer 1-primer 4 performs DNA amplification by using B19 virus nucleic acid template, primer 5-primer 8 performs DNA amplification by using internal standard beta globin gene as template, and the detailed principle is shown in attached figure 1 and figure 3:
primer 1-primer 4 is specifically combined with a B19 virus nucleic acid template, Nfo enzyme meets a tetrahydrofuran modified site in a patrol process and considers that DNA is damaged and needs to be repaired, so the site on a probe is sheared to release a fluorescent group, and the sheared primer 1 has free 3' -OH, so the primer can be used as a primer, under the action of DNA polymerase with a strand displacement function, the primer 1 and the primer 2-primer 4 sheared by Nfo enzyme respectively use one strand in the DNA of B19 virus nucleic acid as a template to carry out primer extension to form a primer extension strand, and thus double-stranded DNA formed by the primer extension strand and the template DNA is obtained.
And under the action of methyl purine DNA glycosylase (AAG) for recognizing and cutting 3-methyl adenine modified base in one strand of the double-stranded DNA, Nfo enzyme-cut 3-methyl adenine modified base in the modified part of the primer 1 and the primer 2 is recognized and cut, so that the double-stranded DNA releases the 3-methyl adenine modified base, and the binding stability of the 3-methyl adenine modified part of the primer 1-the primer 4 and the template DNA is reduced.
③ the primer 3 and the primer 4 enter the position for releasing the 3-methyladenine modified base (namely, the modified part region in the primer 1 and the primer 2), are combined with the template DNA, and under the action of the DNA polymerase with the strand displacement function, the primer 3 and the primer 4 are extended to form the extended strand of the primer 3 and the primer 4, thereby obtaining the double-stranded DNA formed by the extended strand of the primer 3 and the primer 4 and the template DNA, and simultaneously, the primer extended strand in the double-stranded DNA is displaced and released to form the released primer extended DNA single strand.
And (iv) performing the above steps 1 to 2 in a cycle in which the extended strands of the primers 3 and 4 in the double-stranded DNA formed by the extended strands of the primers 3 and 4 and the template DNA obtained in the previous cycle are released, thereby producing a large amount of released primer-extended single-stranded DNA obtained by releasing the primer extended strands in the double-stranded DNA by continuous displacement.
The primer 1 and the primer 2 which are not modified by 3-methyladenine are respectively combined with the released primer extension DNA single strand, Nfo enzyme cuts the position of the primer 1 when meeting the tetrahydrofuran modification position in the inspection process so as to release fluorescent groups, the primer 1 cut by Nfo enzyme has free 3' -OH, so that the primer can be used as a primer, DNA extension reaction is continuously carried out under the action of DNA polymerase with strand displacement function by using the released primer extension DNA single strand as a template under the action of DNA polymerase with strand displacement function so as to form an extension strand of the primer 1 and the primer 2, and simultaneously the released primer extension DNA single strand carries out DNA extension reaction by using the modified parts of the primer 1 and the primer 2 as templates so as to obtain a sequence which is matched with the modified parts of the primer 1 and the primer 2 so as to form an extension strand of the released primer extension DNA single strand, the extended strand of the primer 1, the extended strand of the primer 2 and the released extended strand of the primer extended DNA single strand form a double-stranded DNA. The fluorescence released at this step will be detected by the isothermal detection instrument.
Sixthly, circularly performing the steps 1 to 5. The detection principle of the constant temperature probe method of the internal standard HBB is similar to that of the constant temperature probe method of B19 nucleic acid, except that the fluorescent label of the primer 5 is different from that of the primer 1, thereby realizing the multiple fluorescence PCR.
The fluorescence detection channel is FAM/JOE. The reaction is carried out at normal temperature, 1min/cycle and 40cycles, and other corresponding parameters are set according to used instruments and equipment.
(3) Analysis of results
And automatically storing results after the reaction is finished, analyzing the amplification curves of the detection target and the reference gene respectively, and recording the FAM and JOE respectively observed line type and Ct value.
Quality control: negative control: none of the FAM channels had a Ct value or a Ct > 38 (FIG. 4B). Positive control: the Ct of both FAM and internal standard (JOE) channels is less than or equal to 38 (FIG. 4A). The above requirements need to be met simultaneously in the same experiment, otherwise, the experiment is invalid and needs to be performed again.
And (5) judging a result:
whether the internal standard (JOE) has a typical S-shaped amplification curve or not is analyzed, and if yes, the detection is effective, and subsequent analysis can be continued.
Firstly, if the typical S-type amplification curve is detected by the FAM channel, and Ct is less than or equal to 38, the human parvovirus B19 is positive. If the FAM channel does not detect the typical S-type amplification curve, it indicates that human parvovirus B19 is negative.
Secondly, for positive samples, internal standard detection results do not require; and for a negative sample, detecting the internal standard to be positive, if the internal standard is detected to be negative, detecting the result of the sample to be invalid, searching and eliminating the reason, resampling the sample, and performing repeated tests.
Verification example 1
This example evaluates the stability of the lyophilized reagents provided in example 2, with the following specific experimental procedures and results:
the stability of the lyophilized reagent provided in example 2 was evaluated by storing the lyophilized reagent at room temperature for half a year and 1 year, and a control group was set, in which the lyophilized reagent was stored at room temperature for half a year and 1 year without adding a lyoprotectant. At the same time, the detection limit reference (the added concentration of B19 virus is 1 copy/reaction) of the reagent added with the freeze-drying protective agent and the reagent added with no freeze-drying protective agent are respectively detected 20 times. And if the detection rate is more than 95%, the stability of the reagent is considered to meet the requirement. The results of the tests are shown in Table 4 below.
TABLE 4 stability evaluation results
Figure BDA0003418275710000141
From the above results, the lyophilization process adopted by the kit provided in example 2 can meet the stability requirement of the lyophilized reagent, and the lyophilization protectant can effectively prevent the decrease of the activity of the three enzymes, thereby ensuring the stability of the detection reagent.
Verification example 2
This example explores the sensitivity of the multiplex isothermal probe method provided in example 4 for detecting B19 nucleic acid, and the specific experimental methods and results are as follows:
test samples containing pseudoviruses of B19 sequences were prepared (wherein the concentrations of the pseudoviruses of B19 sequences were 10000, 1000, 100, 10, 1 and 0 copies/reaction), and the results of the test were shown in FIG. 5, using the method provided in example 4.
As can be seen from FIG. 5, the sensitivity of the method provided herein for detecting B19 nucleic acid can reach 1 copy/reaction.
In addition, the present inventors compared the method provided by the present invention with the parameters of the existing isothermal amplification technology, and see table 5 for details.
TABLE 5 comparison of the multiplex isothermal Probe method of the present invention with other isothermal nucleic acid amplification techniques
Figure BDA0003418275710000151
In conclusion, the multiple isothermal probe method provided by the invention has significant advantages compared with other isothermal nucleic acid amplification technologies.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> Shanghai university of traffic medical college affiliated renji hospital
<120> a kit and method for detecting human parvovirus B19 integrating sample processing, nucleic acid extraction and multiple isothermal amplification
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 206
<212> DNA
<213> human parvovirus B19 nucleic acid target gene sequence (artificial sequence)
<400> 1
ccactatgaa aactgggcaa taaactacac ttttgatttc cctggaatta atgcagatgc 60
cctccaccca gacctccaaa ccaccccaat tgtcacagac accagtatca gcagcagtgg 120
tggtgaaagc tctgaagaac tcagtgaaag cagctttttt aacctcatca ccccaggcgc 180
ctggaacact gaaaccccgc gctcta 206
<210> 2
<211> 106
<212> DNA
<213> internal Standard HBB target Gene sequence (artificial sequence)
<400> 2
tgaaggctca tggcaagaaa gtgctcggtg cctttagtga tggcctggct cacctggaca 60
acctcaaggg cacctttgcc acactgagtg agctgcactg tgacaa 106
<210> 3
<211> 54
<212> DNA
<213> primer 1 (artificial sequence)
<400> 3
ccnctntgnn nnctgggcna taaactacac ttttgatttc cctggaatta atgc 54
<210> 4
<211> 34
<212> DNA
<213> primer 2 (artificial sequence)
<400> 4
tngngcgcgg ggtttcngtg ttccaggcgc ctgg 34
<210> 5
<211> 19
<212> DNA
<213> primer 3 (artificial sequence)
<400> 5
ccnctntgnn nnctgggcn 19
<210> 6
<211> 17
<212> DNA
<213> primer 4 (artificial sequence)
<400> 6
tngngcgcgg ggtttcn 17
<210> 7
<211> 49
<212> DNA
<213> primer 5 (artificial sequence)
<400> 7
tgnnggctcn tggcnagnaa gtgctcggtg cctttagtga tggcctggc 49
<210> 8
<211> 36
<212> DNA
<213> primer 6 (artificial sequence)
<400> 8
ttgtcncngt gcngctcnct cagtgtggca aaggtg 36
<210> 9
<211> 18
<212> DNA
<213> primer 7 (artificial sequence)
<400> 9
tgnnggctcn tggcnagn 18
<210> 10
<211> 18
<212> DNA
<213> primer 8 (artificial sequence)
<400> 10
ttgtcncngt gcngctcn 18

Claims (10)

1. A primer composition for detecting human parvovirus B19 at normal temperature is characterized by comprising a primer aiming at the nucleotide sequence of human parvovirus B19 and having SEQ ID NO. 3-6 and a primer aiming at the nucleotide sequence of an internal standard beta globin gene and having SEQ ID NO. 7-10, wherein N in the sequences is 3-methyladenine.
2. The primer composition of claim 1, wherein the primers with the nucleotide sequences of SEQ ID NO. 3 and SEQ ID NO. 7 are specific fluorescent probes; tetrahydrofuran modification sites are introduced into the fluorescent probe, and a fluorescent reporter group and a fluorescent quenching group are coupled in the vicinity of the tetrahydrofuran modification sites.
3. The primer composition of claim 2, wherein the primers with the nucleotide sequences of SEQ ID NO. 3 and SEQ ID NO. 7 are:
SEQ ID NO:3：CCNCTNTGNNNNCTGGGCNATAAACTACACTTTTGATTTCCCT(FAM-dT)(THF)(BHQ1-dT)GGAATTAATGC-PO4；
SEQ ID NO:7：TGNNGGCTCNTGGCNAGNAAGTGCTCGGTGCCTTTAGT(JOE-dT)(THF)(BHQ1-dT)GATGGCCTGGC-PO4。
4. a kit for detecting human parvovirus B19, which integrates sample processing, nucleic acid extraction and multiple isothermal amplification, and is characterized by comprising isothermal amplification detection reagents containing the primer composition according to any one of claims 1 to 3, reagents for nucleic acid isolation/purification, a negative control and a positive control;
the constant temperature amplification detection reagent also comprises dNTP and Mg2+Buffer, Bsu DNA polymerase, methylpurine DNA glycosylase and Nfo enzyme;
the reagent for nucleic acid separation/purification includes chelex100 chelating resin, NaOH, sodium dodecyl sulfate and an internal standard HBB linearized plasmid.
5. The kit according to claim 4, wherein the isothermal amplification detection reagent comprises the following components in concentration: 20mM to 50mM NaOH,0.3-0.6% PEG8000, 4-5mM magnesium chloride, 10-30mM Tris-HCl, 30-50mM KCl, 10-20mM ammonium sulfate, 200. mu.M-400. mu.M dATP, 200. mu.M-400. mu.M dGTP, 200. mu.M-400. mu.M dCTP, 100. mu.M-200. mu.M dTTP, 100. mu.M-200. mu.M dUTP, 0.1-0.3. mu.M of each primer, 0.005-0.01U/. mu.L Nfo enzyme, 0.05-0.1U/. mu.L methylpurine DNA glycosylase and 0.3-0.5U/. mu.L Bsu DNA polymerase; preferably, the reagent for nucleic acid isolation/purification includes the following components at final concentrations: 2 to 5 percent of chelex100 chelating resin, 0.2 to 0.5 percent of NaOH, 0.1 to 0.5 percent of sodium dodecyl sulfate and 10 percent of sodium dodecyl sulfate2-103Copies/. mu.L of internal standard HBB linearized plasmid.
6. The kit according to claim 4, wherein the isothermal amplification detection reagent and the reagent for nucleic acid separation/purification are in a lyophilized powder form; the isothermal amplification detection reagent further comprises a freeze-drying protective agent, which comprises: 1% -5% glycogen, 1% -5% bovine serum albumin and 0.1-0.2 ng/. mu.L carrier RNA.
7. The kit according to claim 6, wherein the lyophilized powder form of the isothermal amplification detection reagent is prepared by the following method:
(1) preparation of nucleic acid amplification reagents: adding NaOH, PEG8000, magnesium chloride, Tris-HCl, KCl, ammonium sulfate, calf serum albumin, dATP, dGTP, dCTP, dTTP, dUTP, primers, AAG, Bsu DNA polymerase, Nfo enzyme and sterilized purified water into a centrifugal tube according to a certain proportion, and uniformly mixing;
(2) preparing a freeze-drying protective agent: weighing a certain amount of glycogen and bovine serum albumin, adding the glycogen and the bovine serum albumin into the sterilized purified water for dissolving, adding carrier RNA, and fixing the volume to the final volume;
(3) taking out each constant-temperature amplification reagent and the freeze-drying protective agent according to the volume of 100: (3-5), and subpackaging the mixture into PCR reaction tubes according to the reaction amount of 40 mu L;
(4) and (3) immersing the PCR reaction tube into liquid nitrogen for pre-freezing for 2-5 minutes, and directly putting the PCR reaction tube into a freeze dryer for vacuum freeze drying.
8. A method for detecting human parvovirus B19 by integrating collection sample treatment, nucleic acid extraction and multiple isothermal amplification according to any one of the kits of claims 4 to 7, comprising the steps of:
the method comprises the following steps: releasing single-stranded DNA from a sample to be tested into a reaction tube containing the nucleic acid amplification reagent by centrifugation using the reagent for nucleic acid separation/purification;
placing the reaction tube into a sample groove of an amplification instrument, setting positive control and negative control, and then carrying out amplification reaction;
and step three, automatically storing results after the reaction is finished, and analyzing the amplification curves of the detection target and the reference gene respectively.
9. The method of claim 8, wherein the centrifugation conditions in step one are 5000 rpm for 10 seconds; preferably, the fluorescence detection channel adopted in the second step is FAM/JOE; the condition of the amplification reaction is 10-40 ℃; 1 minute/cycle, 40 cycles.
10. An apparatus for use in the method according to claim 8 or 9, wherein the apparatus is a PCR reaction tube comprising a micro-separation tube, comprising a separation tube containing a reagent for nucleic acid separation/purification at an upper portion and a PCR reaction tube containing a reagent for isothermal amplification at a lower portion; the separation tube is inserted into the lower PCR reaction tube to realize the connection of the two;
the bottom of the separation tube is a filter membrane with the diameter of 0.45 mu m.
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