CN114277112B

CN114277112B - Reagent for direct PCR amplification, detection kit and application

Info

Publication number: CN114277112B
Application number: CN202210205587.4A
Authority: CN
Inventors: 董晋涛; 金捷; 杜赞花; 其他发明人请求不公开姓名
Original assignee: Shanghai Siludi Medical Laboratory Co ltd
Current assignee: Shanghai Siludi Medical Laboratory Co ltd
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2022-05-24
Anticipated expiration: 2042-03-04
Also published as: CN114277112A

Abstract

The invention discloses a reagent for direct PCR amplification, a nucleic acid detection kit and application. The reagent comprises NP40 and sarcosyl, wherein the volume percentage of NP40 in a PCR reaction system is 0.1% -2%, and the volume percentage of sarcosyl in the PCR reaction system is 0.1% -2%. The invention compares different surfactants, finds that the combined use of the sodium dodecyl sarcosinate and the NP40 not only has the function of nucleic acid release, but also does not inhibit PCR reaction, and can better realize the direct PCR amplification of the throat swab sample. The direct PCR amplification reagent provided by the invention can realize the detection of new corona nucleic acid from a sample to a result in half an hour; greatly shortens the detection time of the prior new coronavirus and simplifies the operation flow.

Description

Reagent for direct PCR amplification, detection kit and application

Technical Field

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a reagent for direct PCR amplification, a detection kit and application.

Background

At present, nucleic acid detection kits for realizing rapid detection based on isothermal amplification technology exist in the market, but the overall sensitivity is low, while the conventional fluorescence quantitative PCR detection technology has higher detection sensitivity and specificity, but almost all needs a nucleic acid purification and extraction step or a nucleic acid release step before amplification, and the operation is complicated.

Direct PCR (direct PCR) is a reaction for directly amplifying a biological sample without nucleic acid extraction, and is one of the popular techniques in the field of molecular diagnosis at present. The main difference from the conventional PCR is that the sample can be directly subjected to PCR reaction without nucleic acid extraction, and thus, the tolerance of the enzyme participating in the direct PCR reaction and the compatibility of the buffer are highly required.

In the prior art, although a nucleic acid extraction-free technology, for example, patent 200910309980.2, 202010072514.3 and the like disclose a reagent with a nucleic acid releasing function, there is a common feature that a surfactant SDS is used, but the SDS reagent has a serious inhibition effect on PCR amplification, which may cause a reduction in amplification efficiency of PCR.

Disclosure of Invention

The invention aims to provide a reagent for direct PCR amplification, a detection kit and application. Aims to improve the reaction efficiency of direct PCR amplification in the nucleic acid non-extraction technology.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a reagent for direct PCR amplification, wherein the concentration of each component of the reagent in a PCR reaction system is as follows: 0.1-2% of NP40 by volume, 0.1-2% of sodium dodecyl sarcosinate by volume, 30-50mM of Tris-HCl, 5-10% of maltose, 4-6% of galactitol, 1-3U of Taq enzyme, 5-20U of reverse transcriptase and 5-20U of RNase inhibitor.

As a preferred embodiment, the volume percentage of the sodium lauryl sarcosine in the PCR reaction system is 0.3% -2%.

As a preferred embodiment, the volume percentage of the NP40 in the PCR reaction system is 0.8% -1.2%, and the volume percentage of the sodium lauryl sarcosine in the PCR reaction system is 0.4% -1%.

In a preferred embodiment, the reagent further comprises dNTP 0.4-0.5 mM, Mg²⁺2-5 mM, KCl 30-50mM, ammonium sulfate 5-20 mM, and BSA 0.1wt% -1 wt%.

The dNTPs include dATP, dGTP, dCTP and dTTP, where 0.4-0.5 mM dNTP means 0.4-0.5 mM of dATP, dGTP, dCTP and dTTP respectively

The invention also provides application of the reagent in preparation of a kit for detecting the novel coronavirus.

As a preferred embodiment, the kit detects ORF1ab gene and/or N gene of the new coronavirus, and primers for detecting ORF1ab gene are shown as SEQ ID No. 1-2, and probes are shown as SEQ ID No. 3; the primers for detecting the N gene are shown as SEQ ID NO. 12-13, and the probe is shown as SEQ ID NO. 14.

In a preferred embodiment, the concentration of the primer in the PCR reaction system is 240-400 nM, and the concentration of the probe in the PCR reaction system is 120-200 nM.

As a preferred embodiment, a human target RNase P is used as an internal standard, internal standard primers are shown in SEQ ID NO. 17-18, and probes are shown in SEQ ID NO. 19.

The English abbreviations related to the invention are annotated as follows:

BSA: bovine serum albumin

dNTP: is a general term for dATP, dGTP, dCTP, dTTP

NP 40: ethyl phenyl polyethylene glycol

SDS (sodium dodecyl sulfate): sodium dodecyl sulfate

Brij 35: dodecyl polyglycol ether

Compared with the prior art, the invention has the beneficial effects that:

1, the invention compares different surfactants, and finds that the combined use of the sodium dodecyl sarcosinate and the NP40 not only has the function of nucleic acid release, but also does not inhibit the PCR reaction, and can better realize the direct PCR amplification of the pharynx swab sample.

2, the direct PCR amplification reagent provided by the invention can realize the detection of new corona nucleic acid from a sample to a result in half an hour; greatly shortens the detection time of the prior new coronavirus and simplifies the operation flow.

Drawings

FIG. 1 is a schematic diagram of PCR amplification with virus concentration 8000copies/mL for sensitive detection in example 5 of the present invention;

FIG. 2 is a schematic diagram of PCR amplification with a virus concentration of 800copies/mL for sensitive detection in example 5 of the present invention;

FIG. 3 is a schematic diagram of PCR amplification with a virus concentration of 400copies/mL for sensitive detection in example 5 of the present invention;

FIG. 4 is a schematic diagram of PCR amplification with a virus concentration of 200copies/mL for sensitive detection in example 5 of the present invention;

FIG. 5 is a schematic diagram of PCR amplification with a virus concentration of 100copies/mL for sensitive detection in example 5 of the present invention;

FIG. 6 is a schematic diagram of PCR amplification for repeatedly detecting virus at a concentration of 1200copies/mL in example 7 of the present invention;

FIG. 7 is a schematic diagram of PCR amplification for repeatedly detecting a virus concentration of 600copies/mL in example 7 of the present invention;

FIG. 8 is a schematic diagram showing PCR amplification of a negative pharyngeal swab for repeated detection in example 7 of the present invention;

FIG. 9 is a schematic diagram of PCR amplification for detection of interfering substances in example 8 of the present invention.

Detailed Description

The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified.

Example 1 Rapid detection of New coronavirus by direct PCR amplification reagent

The components of the reagent required by the direct PCR amplification kit are shown in the table 1; the specific components of the RT-PCR reaction system are shown in Table 2. The final concentration in Table 2 means the concentration of each component in the PCR reaction system (the concentration in the total volume of 40. mu.L).

TABLE 1

TABLE 2

Primer Probe sequence screening

Aiming at the characteristics of the nucleic acid sequence of the published novel coronavirus, different target point primer probes including novel coronavirus ORF1ab and N are designed, wherein the ORF1ab gene probe is marked by FAM, and the N gene probe is marked by ROX to detect the novel coronavirus in a same reaction system by double targets, so that the accuracy and reliability of detection are improved, and missing detection is prevented.

In addition, in order to monitor the specimen collection, nucleic acid extraction process and PCR amplification process, a human target RNase P is used as an internal standard, a specific primer probe is designed for the internal standard, the probe is marked by CY5 and is added into a reaction system for PCR reaction, so that false negative caused by kit failure or individual sample misoperation is eliminated.

In the design process of the primers and the probes, the primer and the probes can avoid the formation of hairpin structures, primer internal dimers, primer-primer dimers and mismatches as much as possible. In addition, the specific primer and probe sequences designed above are compared and analyzed by NCBI Blast online database to avoid non-specific binding with other viruses or human genes. In this example, the primer probe sequences of different amplification regions were screened for ORF1ab and N targets detected by the novel coronavirus. Specific primer probe sequences are shown in table 3.

TABLE 3

The new corona clinical sample nucleic acid was detected, diluted to the corresponding test concentrations, and screened for different combinations of ORF1ab primer probes according to the protocol in table 4, respectively:

TABLE 4

The amplification Ct values are shown in Table 5:

TABLE 5

The amplification result shows that the Ct value of the scheme 1 is the minimum, the amplification efficiency is the highest, and the scheme 1 is preferred.

The new crown clinical sample nucleic acid was detected according to the protocol in table 6, diluted to the corresponding test concentration, and subjected to N gene primer probe screening of different combinations:

TABLE 6

The amplified Ct values are shown in table 7:

TABLE 7

The amplification result shows that the Ct value of the scheme 2 is the minimum, the amplification efficiency is the highest, and the scheme 2 is preferred.

The preferred ORF1ab and N gene primer probes and internal reference related primer probes were combined (as shown in table 8) for subsequent testing.

TABLE 8

Sample processing

Samples preserved by the guanidine salt (guanidine hydrochloride or guanidine isothiocyanate) inactivation preservation solution can cause enzyme systems in an RT-PCR system to be inactivated, so that PCR is directly inhibited, and the sample preservation solution applicable to the invention is non-guanidine salt (guanidine hydrochloride or guanidine isothiocyanate) sample preservation solution, including culture base substances (Hank's preservation solution), physiological saline, PBS (phosphate buffer solution) preservation solution and the like.

The sample preserved by the non-guanidinium sample preserving fluid does not need extra treatment, and the sample is directly added into a PCR reaction system for amplification. Specifically, the results are shown in Table 9.

TABLE 9

The PCR amplification procedure is shown in Table 10.

Watch 10

The matched PCR amplification Instrument is a Quantstudio 5 Real-Time PCR Instrument. The interpretation of the amplification results and the determination of the detection results are shown in Table 11.

TABLE 11

Note: "+" indicates positive detection target and a Ct value cut off of 39; "-" indicates negative detection target.

Example 2 comparison of the Effect of different surfactants on direct PCR amplification

The formula of example 1 with surfactant removed (NP 40 and sarcosyl) was used as a basic formula, comparing the effect of commonly used surfactants in the prior art SDS, Brij35 and the sarcosyl screened by the present invention on the direct PCR amplification.

N-gene pseudovirus of SARS-CoV-2 quantified by ddPCR was diluted with a negative pharyngeal swab sample matrix stored in a non-guanidinium salt sample storage solution (Virusoanalog kit manufactured by Youkang Biotechnology, Beijing) Ltd.: MT 0301-1) to 5000copies/mL and 500copies copies/mL, and the test was performed according to the protocol in Table 12.

TABLE 12

Of the four schemes in table 12, scheme 1: no surfactant was added as a control; scheme 2: adding surfactant NP40 and SDS; scheme 3: adding surfactant NP40 and Brij 35; scheme 4: surfactant NP40 and sodium lauryl sarcosinate were added. The results of amplification of SARS-CoV-2 virus N gene by the four protocols are shown in Table 13.

Watch 13

The results show that: the Ct value of the amplified product is significantly delayed after SDS is added, indicating that the amplified product inhibits PCR, relative to the control and other surfactants. The combination with the addition of sarcosyl was superior to the combination with Brij35, and the combination of surfactant NP40 and sarcosyl in case 4 had the best amplification effect.

Example 3 comparison of the Effect of different concentrations of sarcosyl on direct PCR amplification

Based on the surfactant-removed formulation of example 1, different concentrations of sarcosyl were set for the combination of sarcosyl and NP40 of example 2 to compare the effect on PCR amplification.

N-gene pseudovirus of SARS-CoV-2 quantified by ddPCR was diluted with a negative pharyngeal swab sample matrix stored in a non-guanidinium salt sample storage solution (Virusoanalog kit manufactured by Youkang Biotechnology (Beijing) Ltd.: MT 0301-1), diluted to 5000copies/mL and 500copies/mL, and tested according to the protocol in Table 14.

TABLE 14

In the schemes 1 to 4 in Table 14, the surfactant NP40 was found to be 1% by volume in the PCR reaction system, and the surfactant sarcosyl was found to be 0.1%, 0.3%, 0.5%, 2%, and 3% by volume, respectively. The results of SARS-CoV-2 virus N gene amplification with different concentrations of sarcosyl are shown in Table 15.

Watch 15

The results show that for different concentrations of the surfactant lauryl sarcosine sodium, the combination effect of the 31% NP40 and the 0.5% lauryl sarcosine sodium is the best, the Ct value is the smallest, and as the preferred scheme, when the concentration of the lauryl sarcosine sodium is too high, such as 3%, the amplification effect is also poor, and the low-concentration sample cannot be detected.

Example 4 comparison of the Effect of different concentrations of NP40 on direct PCR amplification

Based on the preferred combination of concentrations of sarcosyl in example 3, different concentrations of NP40 were set to compare their effect on PCR amplification.

N-gene pseudovirus of SARS-CoV-2 quantified by ddPCR was diluted with a negative pharyngeal swab sample matrix stored in a non-guanidinium salt sample storage solution (Virusoanalog kit manufactured by Youkang Biotechnology (Beijing) Ltd.: MT 0301-1), diluted to 5000copies/mL and 500copies/mL, and tested according to the protocol in Table 16.

TABLE 16

In the cases 1-4 of Table 16, the volume percentage of sarcosyl in the PCR reaction system was 0.5%, and the volume percentage of NP40 was 0.1%, 1%, 2%, 3%, respectively. The results of SARS-CoV-2 virus N gene amplification by NP40 at various concentrations are shown in Table 17.

TABLE 17

The results show that: the new crown N gene pseudovirus can be effectively amplified by setting different concentrations of surfactant NP40, wherein the combination effect of 1% NP40 in the scheme 2 is the best, and the Ct value is the minimum, and the scheme is taken as a preferred scheme.

Example 5 detection sensitivity

Referring to the preferred PCR amplification combination formulation of example 1 and example 4, the novel coronavirus (SARS-CoV-2) pseudovirus quantified by ddPCR was diluted with negative pharyngeal swabs stored in the medium-based preservation solution to 8000copies/mL, 800copies/mL, 400copies/mL, 200copies/mL, and 100copies/mL for 3-fold detection, which resulted in detection of 200copies/mL, and the detection results are shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4, and FIG. 5; the data are shown in Table 18.

Watch 18

Example 6 detection specificity

The primer probe combination and amplification detection method in example 1, and the preferred PCR reagent combination in example 4 were used to verify the specificity of the overall reagent combination.

In order to prove the cross-reactivity of the kit, 21 pathogens such as human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1, SARS coronavirus, MERS coronavirus, diplococcus meningitides, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae, rubella virus, mumps virus, respiratory adenovirus type 3, respiratory adenovirus type 7, respiratory syncytial virus type B, parainfluenza virus type 2, streptococcus pyogenes, Bordetella pertussis, Candida albicans, Pseudomonas aeruginosa, staphylococcus epidermidis and the like and human genome DNA are selected for detection respectively.

The virus was first diluted to 1E +05 CFU/mL, the bacterial and human genomic DNA was diluted to 1E +06 copies/mL,

then, a fluorescent PCR test was performed (see example 1 for details), and it was examined whether or not the detection could cross-react with the above-mentioned pathogen or human genomic DNA. The reaction results are shown in Table 19.

As can be seen from the reaction results, 21 pathogens and human genome DNA have no cross-reactivity, and the detection results are negative and have good specificity.

Watch 19

Example 7 measurement of reproducibility

The primer probe combination and the amplification detection method in the embodiment 1 and the PCR reagent combination in the embodiment 4 are preferably used for repeated detection.

Diluting the novel coronavirus (SARS-CoV-2) pseudovirus quantified by ddPCR with negative pharyngeal swabs preserved by the culture medium-based preservation solution to 1200copies/mL and 600copies/mL, simultaneously detecting the negative pharyngeal swabs which are marked as R1, R2 and R3, and carrying out 10-hole detection, wherein the amplification result is shown in figure 6, figure 7 and figure 8; the results of the test data are shown in Table 20. The results show that: ct value CV is within 5 percent, and the repeatability is good.

Watch 20

Example 8 detection of interfering substances

The primer probe combination and the amplification detection method in example 1 and the PCR reagent combination preferred in example 4 are used to detect the interfering substance.

2 endogenous interfering substances were selected: normal human whole blood, mucin, and 24 foreign interfering substances commonly used in respiratory tract infections: phenylephrine, oxymetazoline, sodium chloride, beclomethasone, dexamethasone, flunisolide, triamcinolone acetonide, budesonide, mometasone, fluticasone, histamine hydrochloride, interferon-alpha, zanamivir, ribavirin, oseltamivir, peramivir, lopinavir, ritonavir, abidol, levofloxacin, tobramycin, azithromycin, ceftriaxone, meropenem.

Novel coronavirus (SARS-CoV-2) pseudovirus quantified by ddPCR was diluted to 600copies/mL (3 XLOD) with a negative pharyngeal swab stored in a medium-type storage solution, an interfering substance was added to the diluted sample in accordance with the interfering concentration shown in Table 16, fluorescence PCR was carried out with no interfering substance added as a control, each sample was repeatedly assayed 3 times, the results were judged for negative and positive, and the effect on the assay results before and after the addition of each interfering substance was evaluated. As can be seen from the following statistical results, the samples added with 2 endogenous sampling interfering substances and 24 drugs do not affect the judgment of negative and positive compared with each control, and the amplification results are shown in FIG. 9.

TABLE 21

The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.

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Claims

1. A reagent for direct PCR amplification, the reagent comprising the components: NP40, dodecyl sarcosine sodium, Tris-HCl, maltose, galactitol, Taq enzyme, reverse transcriptase, RNase inhibitor; the concentration of each component in the PCR reaction system is as follows: 0.1-2% of NP40 by volume, 0.1-2% of sodium dodecyl sarcosinate by volume, 30-50mM of Tris-HCl, 5-10% of maltose, 4-6% of galactitol, 1-3U of Taq enzyme, 5-20U of reverse transcriptase and 5-20U of RNase inhibitor.

2. The reagent for direct PCR amplification according to claim 1, wherein: the volume percentage of the sodium dodecyl sarcosinate in a PCR reaction system is 0.3-2%.

3. The reagent for direct PCR amplification according to claim 1, wherein: the volume percentage of the NP40 in the PCR reaction system is 0.8-1.2%, and the volume percentage of the sarcosyl in the PCR reaction system is 0.4-1%.

4. The reagent for direct PCR amplification according to claim 1, wherein: the reagent also comprises dNTP 0.4-0.5 mM and Mg²⁺2-5 mM, KCl 30-50mM, ammonium sulfate 5~20mM、BSA 0.1wt%~1wt%。

5. Use of a reagent according to any one of claims 1 to 4 in the manufacture of a kit for detecting a novel coronavirus.

6. Use of the reagent according to claim 5 for the preparation of a kit for the detection of a novel coronavirus, characterized in that: the kit is used for detecting ORF1ab gene of ORF1ab gene of new coronavirus, wherein the primer is shown as SEQ ID NO. 1-2, and the probe is shown as SEQ ID NO. 3.

7. Use of the reagent according to claim 5 for the preparation of a kit for the detection of a novel coronavirus, characterized in that: the kit detects the N gene of the new coronavirus, and a primer for detecting the N gene is shown as SEQ ID No. 12-13, and a probe is shown as SEQ ID No. 14.

8. Use of the reagent according to claim 6 or 7 for the preparation of a kit for the detection of a new coronavirus, characterized in that: the concentration of the primer in a PCR reaction system is 240-400 nM, and the concentration of the probe in the PCR reaction system is 120-200 nM.

9. Use of the reagent according to claim 6 or 7 for the preparation of a kit for the detection of a new coronavirus, characterized in that: a human target RNase P is used as an internal standard, internal standard primers are shown as SEQ ID NO. 17-18, and probes are shown as SEQ ID NO. 19.