KIT FOR CALIBRATION OF ISOTHERMAL POLYMERASE CHAIN REACTION (PCR) ANALYZER AND USE THEREOF

Abstract

Provided is a kit for calibration of an isothermal polymerase chain reaction (PCR) analyzer and use thereof. The kit includes a standard substance, an amplification primer set, a reaction buffer, a polymerase, a dye, and a negative control. The standard substance is a DNA plasmid with a gradient concentration of 10.sup.0 copies/L to 10.sup.6 copies/L; the amplification primer set has nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6; the polymerase is a Bst DNA polymerase; and the dye is a loop-mediated isothermal amplification (LAMP) fluorescent dye (with excitation: 485 nm, emission: 498 nm, and a detection channel of SYBR Green I or a FAM channel). The kit and a technology for calibration of an isothermal PCR instrument meet calibration demands of the isothermal PCR instrument and fill a technical gap.

Claims

1. A primer set for calibration of an isothermal polymerase chain reaction (PCR) analyzer, wherein the primer set comprises the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6.

2. A kit for calibration of an isothermal PCR analyzer, comprising the primer set according to claim 1.

3. The kit according to claim 2, further comprising a standard substance, a reaction buffer, a polymerase, and a dye.

4. The kit according to claim 3, wherein the standard substance is a DNA plasmid with a gradient concentration of 10.sup.0 copies/L to 10.sup.6 copies/L.

5. The kit according to claim 4, wherein the DNA plasmid is a pMD18 plasmid with a specific sequence shown in SEQ ID NO: 13.

6. The kit according to claim 5, wherein the reaction buffer comprises the following components: 20 mM of Tris-HCl, 10 mM of (NH.sub.4).sub.2SO.sub.4, 50 mM of KCl, 8 mM of MgSO.sub.4, 0.1% of Tween 20, and 1.4 mM each of dNTPs; and the polymerase is a Bst DNA polymerase.

7. The kit according to claim 6, further comprising a negative control.

8. A loop-mediated isothermal amplification (LAMP) reaction system for calibration of an isothermal PCR instrument, wherein the LAMP reaction system has a volume of 25 L and comprises the following components: 12.5 L of 2 reaction buffer, 1 L of Bst DNA polymerase, 0.5 L of 50LAMP dye, 5 pmol of SEQ ID NO: 1, 5 pmol of SEQ ID NO: 2, 40 pmol of SEQ ID NO: 3, 40 pmol of SEQ ID NO: 4. 10 pmol of SEQ ID NO: 5, 10 pmol of SEQ ID NO: 6, and 1 L of the standard substance according to claim 5, and ultrapure water added to make up to 25 L.

9. A method for calibrating an isothermal PCR analyzer, comprising: designing the primer set according to claim 1 and applying the primer set in the process of calibration of the isothermal PCR analyzer.

10. A method for calibrating an isothermal PCR analyzer, comprising applying the kit according to claim 2 in the process of calibration of the isothermal PCR analyzer.

11. The method according to claim 10, wherein the kit further comprises a standard substance, a reaction buffer, a polymerase, and a dye.

12. The method according to claim 11, wherein the standard substance is a DNA plasmid with a gradient concentration of 10.sup.0 copies/L to 10.sup.6 copies/L.

13. The method according to claim 12, wherein the DNA plasmid is a pMD18 plasmid with a specific sequence shown in SEQ ID NO: 13.

14. The method according to claim 13, wherein the reaction buffer comprises the following components: 20 mM of Tris-HCl, 10 mM of (NH.sub.4).sub.2SO.sub.4, 50 mM of KCl, 8 mM of MgSO.sub.4, 0.1% of Tween 20, and 1.4 mM each of dNTPs; and the polymerase is a Bst DNA polymerase.

15. The method according to claim 14, wherein the kit further comprises a negative control.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows a schematic diagram of the kit for calibration of isothermal PCR in the present disclosure;

[0029] FIG. 2 shows amplification effects of 3 sets of base primers in a LAMP repeated experiment in Example 1;

[0030] FIG. 3 shows an amplification effect of a primer set in a LAMP repeated experiment in Example 2;

[0031] FIG. 4 shows LAMP amplification effects at different concentrations in Example 4;

[0032] FIG. 5 shows a measurement range of the LAMP reaction in Example 4; and

[0033] FIG. 6 shows a measurement range of the LAMP reaction for an isothermal PCR instrument in Example 5.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] In the present disclosure, base primers and loop primers are designed based on optimization of Tm value, dG value, GC content, and distance between primers of a target sequence of the standard substance using LAMP and PrimerExplorer V5 software. The standard substance, amplification primers, amplification reagents, and water are configured into a 25 L LAMP reaction system to allow isothermal amplification at 65 C., so as to obtain an exponential amplification inflection point time (min) within 40 min.

[0035] The kit for calibration of isothermal PCR (FIG. 1) includes:

TABLE-US-00002 TABLE 1 No. Component 1 Standard substances (S1, S2, S3, S4, S5, S6, S7) 2 Negative control (TE buffer (10 mM, pH = 7.8)) 3 Amplification primer set 4 2 reaction buffer 5 Bst DNA polymerase 6 50 LAMP dye
(1) Standard substance:

[0036] The standard material is plasmid DNA, and its characteristic value is accurately determined using digital PCR, with a concentration of (10.sup.0-10.sup.6) copies/L. The standard material is used as a standard in the kit for calibration of parameters such as sensitivity and repeatability of an isothermal PCR instrument.

Amplification Primer Set:

TABLE-US-00003 TABLE2 SequenceID Primer No. name Nucleotidesequence SEQIDNo:1 F3 CTGGGTGAAAGGAGACTGT SEQIDNo:2 B3 AGACAGGGCTGTGTTGGC SEQIDNo:3 FIP TGAGAGGGAAATGATTTCAGGACATGAGGGTTACCCC TCGG SEQIDNO:4 BIP GGCATGGAGTCCTCACCCATCGTACAGGTCTTTGCGG ATGT SEQIDNO:5 LF TAGCTTAATCAGGAGAGCC SEQIDNO:6 LB ACCTTCTTCTCCATCATGAAGTG

(2) Reaction System (25 L):

TABLE-US-00004 TABLE 3 Component Working concentration Volume (L) Reaction buffer 1 12.5 Bst DNA polymerase 320 U/mL 1 LAMP dye 1 0.5 F3 200 nM 1 B3 200 nM 1 FIP 1600 nM 1 BIP 1600 nM 1 LF 400 nM 0.25 LB 400 nM 0.25 Standard substance / 1 TRIS EDTA (TE) 0.1 / 5.5 Total system 25

(3) Reaction Program:

[0037] The reaction is maintained at a constant temperature of 65 C. for 60 min. In the prior art, base primers and loop primers are designed based on optimization of Tm value, dG value, GC content, and distance between primers using LAMP and PrimerExplorer V5 software. The standard substance, amplification primers, amplification reagents, and water are configured into a 25 L LAMP reaction system to allow isothermal amplification at 65 C., so as to obtain an exponential amplification inflection point time (min) within 40 min.

[0038] The PCR instrument used in all the following examples is a Roche LightCycler 48011 fluorescence quantitative PCR instrument.

Example 1 Design and Optimization of Base Primers

[0039] In the present disclosure, a primer of the LAMP system was designed for a specific sequence region of a DNA sequence of a pMD18-T plasmid. The base primers (F3, B3, FIP, BIP) were designed using PrimerExplorer V5 software, and the base primers were obtained by optimizing the Tm value, dG value, GC content, and distance between primers.

[0040] The specific sequence was as follows:

TABLE-US-00005 SEQIDNO:13: ACTCTTCCAGCCTTCCTTCCTGGGTGAGTGGAGACTGTAAT CTTGGCTCACCCTCATGAGGGTTACCCCTCGGGGCTGTGCTGTGGAAGC TATCTCCTGCCCTCATTTCCCTCTCAGGCATGGAGTCCTGTGGCATCCAC GTTTCTACCTTCAACTCCATCATGAAGTGTCTGGTGGACATCCGCAAAGA CCTGTACGCAACACAGTGCTGTCTCCGCGCACCACCATCCAAGGGGTGGC ATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCAC CCAGCACAATCTTGATCTTG.

[0041] A DNA plasmid was used as a template, and its copy number concentration was diluted to 10.sup.2 copies/L, and a reaction system was configured according to the composition in Table 4:

TABLE-US-00006 TABLE 4 Component Working concentration Volume (L) Reaction buffer 1 12.5 Bst DNA polymerase 320 U/mL 1 LAMP dye 1 0.5 F3 200 nM 1 B3 200 nM 1 FIP 1600 nM 1 BIP 1600 nM 1 Standard substance / 1 TE 0.1 (TE buffer, including / 6 EDTA 0.1 mM and Tris 10 mM by concentration) Total system 25

[0042] A temperature cycle process of the LAMP reaction was shown in Table 5:

TABLE-US-00007 TABLE 5 Temperature ( C.) Time Number of cycles Isothermal amplification 65 1 min 60

[0043] Readings during LAMP amplification were obtained in real time based on the Roche LightCycler 48011 fluorescence quantitative PCR instrument. In the reaction system of the base primer set, there was obvious amplification of template DNA. The exponential amplification inflection point time (min) automatically read by the instrument is shown in Table 6 to Table 9 while the amplification results are shown in FIG. 2:

TABLE-US-00008 TABLE 6 Exponential amplification inflection point time (min) Base primer set 1 19.65

[0044] The experiment was repeated with the base primer set, and the amplification effect is shown in FIG. 2, with desirable repeatability.

TABLE-US-00009 TABLE7 Sequencesofbaseprimerset1 Primername Sequence F3(SEQIDNO:1) CTGGGTGAAAGGAGACTGT B3(SEQIDNO:2) AGACAGGGCTGTGTTGGC FIP(SEQIDNO:3) TGAGAGGGAAATGATTTCAGGACATGAGGGTTACCCCTCGG BIP(SEQIDNO:4) GGCATGGAGTCCTCACCCATCGTACAGGTCTTTGCGGATGT

TABLE-US-00010 TABLE8 Baseprimerset2 (exponentialamplificationinflectionpointtime(min):21.98) Primername Sequence F3(SEQIDNO:5) ATGAGGGTTACCCCTCGG B3(SEQIDNO:6) TGCATCCTGTCGGCAATG FIP(SEQIDNO:7) GCCACAGGACTCCATGCCTGGCTGTGCTGTGGAAGCTAAG BIP(SEQIDNO:8) TGAAGTGTGACGTGGACATCCGCAGGGTACATGGTGGTGC

TABLE-US-00011 TABLE9 Baseprimerset3(noamplificationachieved) Primername Sequence F3(SEQIDNO:9) ACTCTTCCAGCCTTCCTTCC B3(SEQIDNO:10) GCGTACAGGTCTTTGCGG FIP(SEQIDNO:11) TAGCTTCCACAGCACAGCCCTGGGTGAGTGGAGACTGTC BIP(SEQIDNO:12) TTCCCTCTCAGGCATGGAGTCCTGTCCACGTCACACTTCATG

Example 2 Design and Optimization of Loop Primers

[0045] In the present disclosure, a primer of the LAMP system was designed for a specific sequence region of a DNA sequence of a plasmid. The base primers (F3, B3, FIP, BIP) were designed using PrimerExplorer V5 software, and the loop primer set was obtained by optimizing the Tm value, dG value, GC content, and distance between primers.

[0046] pMD18-T plasmid; the specific sequence was as follows:

TABLE-US-00012 SEQIDNO:13: ACTCTTCCAGCCTTCCTTCCTGGGTGAGTGGAGACTGTAAT CTTGGCTCACCCTCATGAGGGTTACCCCTCGGGGCTGTGCTGTGGAAGC TATCTCCTGCCCTCATTTCCCTCTCAGGCATGGAGTCCTGTGGCATCCAC GTTTCTACCTTCAACTCCATCATGAAGTGTCTGGTGGACATCCGCAAAGA CCTGTACGCAACACAGTGCTGTCTCCGCGCACCACCATCCAAGGGGTGGC ATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCAC CCAGCACAATCTTGATCTTG.

[0047] A DNA plasmid was used as a template, and its copy number concentration was diluted to 10.sup.2 copies/L, and a reaction system was configured according to the composition in Table 10:

TABLE-US-00013 TABLE 10 Component Working concentration Volume (L) Reaction buffer 1 12.5 Bst DNA polymerase 320 U/mL 1 LAMP dye 1 0.5 F3 200 nM 1 B3 200 nM 1 FIP 1600 nM 1 BIP 1600 nM 1 LF 400 nM 0.25 LB 400 nM 0.25 Standard substance / 1 TE 0.1 / 5.5 Total system 25

[0048] A temperature cycle process of the LAMP reaction was shown in Table 11:

TABLE-US-00014 TABLE 11 Temperature ( C.) Time Number of cycles Isothermal amplification 65 1 min 60

[0049] Readings were obtained after LAMP amplification was completed. In all reaction systems containing base primers and loop primers, there was obvious amplification of template DNA, and the exponential amplification inflection point time (min) automatically read by the instrument was shown in Table 12 to Table 15:

TABLE-US-00015 TABLE 12 Exponential amplification inflection point time (min) LAMP 1-1 primer set (including 12.26 base primers and loop primers)

[0050] The experiment was repeated, and the amplification effect was shown in FIG. 3, with desirable repeatability.

TABLE-US-00016 TABLE13 SequencesofLAMP1-1primerset Primername Sequence F3(SEQIDNO:1) CTGGGTGAAAGGAGACTGT B3(SEQIDNO:2) AGACAGGGCTGTGTTGGC FIP(SEQIDNO:3) TGAGAGGGAAATGATTTCAGGACATGAGGGTTACCCCTCGG BIP(SEQIDNO:4) GGCATGGAGTCCTCACCCATCGTACAGGTCTTTGCGGATGT LF(SEQIDNO:14) TAGCTTAATCAGGAGAGCC LB(SEQIDNO:15) ACCTTCTTCTCCATCATGAAGTG

TABLE-US-00017 TABLE14 1-2primerset (exponentialamplificationinflectionpointtime(min):16.87) Primername Sequence F3(SEQIDNO:1) CTGGGTGAAAGGAGACTGT B3(SEQIDNO:2) AGACAGGGCTGTGTTGGC FIP(SEQIDNO:3) TGAGAGGGAAATGATTTCAGGACATGAGGGTTACCCCTCGG BIP(SEQIDNO:4) GGCATGGAGTCCTCACCCATCGTACAGGTCTTTGCGGATGT LF(SEQIDNO:16) TTAGCTTCCACAGCACAGCC LB(SEQIDNO:17) TTCAACTCCATCATGAAGTGTGAC

TABLE-US-00018 TABLE15 1-3primerset (exponentialamplificationinflectionpointtime(min):22.75) Primername Sequence F3(SEQIDNO:1) CTGGGTGAAAGGAGACTGT B3(SEQIDNO:2) AGACAGGGCTGTGTTGGC FIP(SEQIDNO:3) TGAGAGGGAAATGATTTCAGGACATGAGGGTTACCCCTCGG BIP(SEQIDNO:4) GGCATGGAGTCCTCACCCATCGTACAGGTCTTTGCGGATGT LB(SEQIDNO:18) CTCCATCATGAAGTGTGACGTG

Example 3 Optimization of Amplification Temperature

[0051] In this example, the amplification temperature of the LAMP reaction was optimized, and an amplification temperature gradient was set to: 64 C., 65 C., and 66 C.

[0052] A DNA plasmid was used as a template, and its copy number concentration was diluted to 10.sup.2 copies/L, and a reaction system was configured using primers shown in SEQ ID NO: 1 to SEQ ID NO: 6 according to the composition in Table 16:

TABLE-US-00019 TABLE 16 Component Working concentration Volume (L) Reaction buffer 1 12.5 Bst DNA polymerase 320 U/mL 1 LAMP dye 1 0.5 F3 200 nM 1 B3 200 nM 1 FIP 1600 nM 1 BIP 1600 nM 1 LF 400 nM 0.25 LB 400 nM 0.25 Standard substance / 1 TE 0.1 / 5.5 Total system 25

[0053] The temperature cycle process of the LAMP reaction is shown in Table 17:

TABLE-US-00020 TABLE 17 Temperature ( C.) Time Number of cycles Isothermal amplification 64/65/66 1 min 60

[0054] Readings were obtained after LAMP amplification was completed. In reaction systems of 64 C./65 C./66 C., there was obvious amplification of template DNA, and the exponential amplification inflection point time (min) automatically read by the instrument is shown in Table 18:

TABLE-US-00021 TABLE 18 Exponential amplification Temperature ( C.) inflection point time (min) 64 16.29 65 12.26 66 14.28

[0055] Based on this, the amplification temperature of the reaction system was finally determined to be 65 C.

Example 4 Determination for Measurement Range of LAMP Reaction

[0056] A DNA plasmid was used as a template, and its copy number concentration was gradient diluted to (10.sup.0-10.sup.6) copies/L, and a reaction system was configured using primers shown in SEQ ID NO: 1 to SEQ TD NO: 6 according to the composition in Table 19:

TABLE-US-00022 TABLE 19 Component Working concentration Volume (L) Reaction buffer 1 12.5 Bst DNA polymerase 320 U/mL 1 LAMP dye 1 0.5 F3 200 nM 1 B3 200 nM 1 FIP 1600 nM 1 BIP 1600 nM 1 LF 400 nM 0.25 LB 400 nM 0.25 Standard substance / 1 TE 0.1 / 5.5 Total system 25

[0057] The temperature cycle process of the LAMP reaction is shown in Table 20:

TABLE-US-00023 TABLE 20 Temperature ( C.) Time Number of cycles Isothermal amplification 65 1 min 60

[0058] LAMP was completed and readings were obtained, and the amplification effect is shown in FIG. 4. After repeated experiments, an average exponential amplification inflection point time (min) (FIG. 5) is shown in Table 21:

TABLE-US-00024 TABLE 21 Exponential amplification inflection DNA point time (min) concentration Average (copies/L) 1 2 3 value RSD 10.sup.6 6.00 6.36 6.00 6.12 3.40% 10.sup.5 7.21 7.17 7.11 7.16 0.70% 10.sup.4 8.63 8.66 8.7 8.66 0.41% 10.sup.3 10.03 10.14 9.88 10.02 1.30% 10.sup.2 12.26 14.36 13.82 13.48 8.09% 10.sup.1 15.06 15.28 15.28 15.21 0.84% 10.sup.0 36.51 35.34 35.34 35.73 1.89%

[0059] Therefore, the reaction measurement of this kit for calibration of an isothermal PCR instrument ranged from (10.sup.0-10.sup.6) copies/L.

Example 5 Example for Calibration of Isothermal Amplification PCR Instrument

[0060] The isothermal PCR instrument was calibrated based on the kit and method of the present disclosure, and a calibration system was a reaction system configured according to the following Table 22.

TABLE-US-00025 TABLE 22 Component Working concentration Volume (L) Reaction buffer 1 12.5 Bst DNA polymerase 320 U/mL 1 LAMP dye 1 0.5 F3 200 nM 1 B3 200 nM 1 FIP 1600 nM 1 BIP 1600 nM 1 LF 400 nM 0.25 LB 400 nM 0.25 Standard substance / 1 TE 0.1 / 5.5 Total system 25

[0061] The temperature cycle process of the LAMP reaction is shown in Table 23:

TABLE-US-00026 TABLE 23 Temperature ( C.) Time Number of cycles Isothermal amplification 65 1 min 60

[0062] Readings were obtained after the LAMP amplification was completed. After repeated experiments, an average exponential amplification inflection point time (min) (FIG. 6) is shown in Table 24.

TABLE-US-00027 TABLE 24 Exponential amplification DNA concentration (copies/L) inflection point time (min) 10.sup.3 12.32 10.sup.2 14.38 10.sup.1 25.26 10.sup.0 40.20

Calibration Results:

[0063] (1) sensitivity: 10.sup.0 copies/L; (2) measurement range: 10.sup.0 copies/L to 103 copies/L; (3) measurement repeatability.

[0064] The above examples are only intended to describe the preferred implementations of the present disclosure, but not to limit the scope of the present disclosure. Various alterations and improvements made by those of ordinary skill in the art based on the technical solution of the present disclosure without departing from the design spirit of the present disclosure shall fall within the scope of the appended claims of the present disclosure.