Primer for reducing LAMP non-specific amplification and LAMP method

Abstract

The invention relates to a primer for reducing LAMP (loop-mediated isothermal amplification) nonspecific amplification and an LAMP method. The primer for reducing LAMP non-specific amplification is a closed primer and is completely or partially complementary with an F1c fragment in an upstream internal primer and / or a B1c fragment in a downstream internal primer in the internal primer pair in the LAMP primer group, and the 3'end of the closed primer is modified by a C3 intermediate arm.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to primers for reducing non-specific amplification of LAMP and a LAMP method. Background Art

[0002] The loop-mediated isothermal amplification (LAMP) technology has the characteristics of high sensitivity and rapid response. It can meet the requirements of the World Health Organization for affordability, sensitivity, user-friendliness, rapid / robustness, no equipment required, and easy delivery. Therefore, this technology is widely used in the detection of pathogens in various environmental conditions. However, users have found that this technology has always had the problem of nonspecific amplification, which seriously affects the reliability of the test results. The most typical example is that the false positive rate of the FDA-approved LAMP method for cytomegalovirus detection was significantly higher than the claimed data when it was used in two clinical institutions. It can be seen that establishing a method that can improve the specificity of the amplification process, maintain the sensitivity of the original technology, and is universal is the key to vigorously promote the LAMP technology to obtain reliable results and credible conclusions in daily environments and clinical settings. Summary of the invention

[0003] One of the present inventions provides a blocking primer for reducing non-specific amplification of LAMP, which is completely complementary or partially complementary to the F1c fragment in the upstream internal primer and / or the B1c fragment in the downstream internal primer in the internal primer pair in the LAMP primer set, and has a C3 spacer modification at its 3' end.

[0004] In a specific embodiment, when the blocking primer is partially complementary to the F1c fragment and / or the B1c fragment, the blocking primer lacks 3 to 6 bases complementary to the 5' end of the F1c fragment and / or the B1c fragment.

[0005] In a specific embodiment, when the blocking primer is partially complementary to the F1c fragment and / or the B1c fragment, the blocking primer lacks 3 bases complementary to the 5' end of the F1c fragment and / or the B1c fragment.

[0006] In a specific embodiment, for the UL54 gene of human cytomegalovirus, the sequence of the blocking primer is at least one of SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20; for the UL75 gene of human cytomegalovirus, the sequence of the blocking primer is at least one of SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24.

[0007] The second aspect of the present invention provides a LAMP method for reducing non-specific amplification of LAMP, characterized in that: 1) preparing a LAMP amplification reaction system: the LAMP amplification reaction system at least includes a LAMP primer set, a blocking primer as described in any one of the present inventions, a DNA sample for detection, dNTP, a LAMP fluorescent dye, Mg 2+ , an enzyme for LAMP amplification and a buffer for LAMP amplification; wherein the LAMP primer set consists of an internal primer pair, an external primer pair and a loop primer pair; 2) LAMP amplification reaction: Incubate at 65 degrees Celsius for 50 to 90 minutes, detect the fluorescence signal during the reaction, and then terminate the reaction.

[0008] In a specific embodiment, in step 2), the incubation is carried out at a constant temperature of 65 degrees Celsius for 50 to 75 minutes.

[0009] In a specific embodiment, in step 2), the incubation is carried out at a constant temperature of 65 degrees Celsius for 60 to 70 minutes.

[0010] In a specific embodiment, in step 2), the activity of the enzyme used for LAMP amplification is inactivated at above 95 degrees Celsius to terminate the reaction.

[0011] Beneficial effects of the present invention: The present invention finds that adding a blocking primer that is completely complementary or partially complementary to the F1c fragment in the upstream internal primer and / or the B1c fragment in the downstream internal primer in the internal primer pair in the LAMP primer set can effectively reduce the non-specific amplification of LAMP, thereby reducing false positives. Furthermore, the present invention also finds that appropriately shortening the length of the blocking primer can shorten the detection time of LAMP amplification while ensuring that the non-specific amplification of LAMP is effectively reduced and false positives are reduced, that is, the sensitivity of LAMP amplification is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The LAMP detection analysis without adding DNA template in Example 1 is shown. The amplification curve of each gene is repeated 8 times, and the red dotted line is the baseline threshold of the fluorescence signal detection.

[0013] Figure 2 The figure shows the LAMP detection analysis using a positive DNA template in Example 2. The amplification curve of each treatment is the average result of 8 repeated experiments, and the red dotted line is the baseline threshold of the fluorescence signal detection.

[0014] Figure 3 A schematic diagram of the designed blocking primer F1 or B1 is shown.

[0015] Figure 4 The LAMP detection analysis of each treatment group in Example 3 is shown. Among them, Figure A and Figure C are amplification curves, and the red dotted line is the baseline threshold of fluorescence signal detection; Figure B and Figure D are the average time for the fluorescence signal to reach the baseline threshold (5000RFU) in 8 repeated experiments. The numbers on the bar graph represent the average of the results obtained in 8 repeated experiments. No signal represents a negative result. A one-way ANOVA was performed between each treatment group with a fluorescence signal and the primer control group. **** P <0.0001.

[0016] Figure 5 The figure shows the LAMP detection analysis of each treatment group in Example 4. Among them, Figure A and Figure C are amplification curves, and the red dotted line is the baseline threshold of fluorescence signal detection; Figure B and Figure D are the average time for the fluorescence signal to reach the baseline threshold (5000RFU) in 8 repeated experiments, and the numbers on the bar graph represent the average of the results obtained in 8 repeated experiments. **** indicates that the difference between each treatment group and the primer control group in one-way analysis of variance is P < 0.0001.

[0017] Figure 6 The LAMP detection analysis of each treatment group in Example 5 is shown. The numbers on the bar graph represent the average time for the fluorescence signal to reach the baseline threshold (5000RFU) in 8 repeated experiments. No signal means that no fluorescence signal was detected within 130 minutes. A one-way ANOVA was performed between each treatment group with a fluorescence signal and the primer control group. *** P <0.001, **** P < 0.0001.

[0018] Figure 7The figure shows the LAMP assay analysis of each treatment group in Example 6. The numbers on the bar graph represent the average time for the fluorescence signal to reach the baseline threshold (5000RFU) in 8 repeated experiments. A one-way ANOVA was performed between each treatment group and the primer control group. ***P<0.001, ****P<0.0001, and ns indicates no significant difference. DETAILED DESCRIPTION

[0019] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0020] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0021] In the present invention, the upstream internal primer in the internal primer pair in the LAMP primer set is named with FIP+gene name, FIP and the gene name are connected by a horizontal bar, and it is composed of two sections before and after F1c and F2; the downstream internal primer is named with BIP+gene name, BIP and the gene name are connected by a horizontal bar, and it is composed of two sections before and after B1c and B2. The F1c and B1c parts are used to trigger rapid amplification by forming a dumbbell-shaped structure by folding back during the amplification reaction; the F2 and B2 parts are respectively complementary to the bases of the target gene. The external primer pair is named with F3+gene name and B3+gene name, wherein F3 and the gene name or B3 and the gene name are connected by a horizontal bar. The loop primer pair is named with LF+gene name and LB+gene name, wherein LF and the gene name or LB and the gene name are connected by a horizontal bar. The blocking primer complementary to the F1c portion of the upstream internal primer is named F1+gene name, with F1 and the gene name connected by a horizontal bar. Specifically, when the 3' end of the blocking primer is modified with a C3 spacer and is completely complementary to the F1c portion of the upstream internal primer, it is named F1-SpC3+gene name, with F1-SpC3 and the gene name connected by a horizontal bar; when the 3' end of the blocking primer is modified with a C3 spacer and lacks three bases complementary to the F1c at the 5' end, it is named F1-3C3P+gene name, with F1-3C3P and the gene name connected by a horizontal bar; when the 3' end of the blocking primer is modified with a C3 spacer and lacks six bases complementary to the F1c at the 5' end, it is named F1-3C6P+gene name, with F1-3C6P and the gene name connected by a horizontal bar. The blocking primer B1 which is complementary to the B1c part in the downstream internal primer is named as B1+gene name, and B1 and the gene name are connected by a horizontal bar. Specifically, when the 3' end of the blocking primer is modified with a C3 spacer and is completely complementary to the B1c part in the upstream internal primer, it is named as B1-SpC3+gene name, and B1-SpC3 and the gene name are connected by a horizontal bar; when the 3' end of the blocking primer is modified with a C3 spacer and lacks 3 bases which are complementary to B1c at the 5' end, it is named as B1-3C3P+gene name, and B1-3C3P and the gene name are connected by a horizontal bar; when the 3' end of the blocking primer is modified with a C3 spacer and lacks 6 bases which are complementary to B1c at the 5' end, it is named as B1-3C6P+gene name, and B1-3C6P and the gene name are connected by a horizontal bar. Example 1: LAMP assay without DNA template

[0022] In order to illustrate the beneficial effects of the present invention, human cytomegalovirus genes are selected as the cause for analyzing non-specific amplification of LAMP. Specifically, two target genes are taken as examples, namely UL54 (US NCBI database sequence number: 3077501) and UL75 (US NCBI database sequence number: 3077450).

[0023] The full-length nucleic acid sequences encoding the target genes UL54 and UL75 were obtained from the NCBI public database, and LAMP primers were designed and primer sets for LAMP were generated using the online software PrimerExplorer.

[0024] For the UL54 gene, the sequence information of its primer set is as follows: the upstream internal primer FIP-UL54 sequence is shown in SEQ ID No. 1; the upstream external primer F3-UL54 sequence is shown in SEQ ID No. 2; the upstream loop primer LF-UL54 sequence is shown in SEQ ID No. 3; the downstream internal primer BIP-UL54 sequence is shown in SEQ ID No. 4; the downstream external primer B3-UL54 sequence is shown in SEQ ID No. 5; and the downstream loop primer LB-UL54 sequence is shown in SEQ ID No. 6.

[0025] For the UL75 gene, the sequence information of its primer set is as follows: the upstream internal primer FIP-UL75 sequence is shown in SEQ ID No. 7; the upstream external primer F3-UL75 sequence is shown in SEQ ID No. 8; the upstream loop primer LF-UL75 sequence is shown in SEQ ID No. 9; the downstream internal primer BIP-UL75 sequence is shown in SEQ ID No. 10; the downstream external primer B3-UL75 sequence is shown in SEQ ID No. 11; the downstream loop primer LB-UL75 sequence is shown in SEQ ID No. 12.

[0026] A standard reaction system was established: the total reaction volume was 25 μL, including: 1× isothermal amplification buffer (New England Biolabs), magnesium sulfate solution (MgSO4) with a final concentration of 8 mmol / L, deoxynucleoside triphosphate (dNTP) with a final concentration of 1.4 mmol / L, 50× LAMP fluorescent dye (New England Biolabs) with a final concentration of 0.5 μmol / L, 320 units / mL Bst 2.0 enzyme (New England Biolabs), upstream external primers with a final concentration of 0.2 μmol / L, downstream external primers with a final concentration of 0.2 μmol / L, upstream internal primers with a final concentration of 1.6 μmol / L, downstream internal primers with a final concentration of 1.6 μmol / L, upstream loop primers with a final concentration of 0.4 μmol / L, and downstream loop primers with a final concentration of 0.4 μmol / L, and ultrapure water. It should be noted that in this embodiment, no DNA template was added to the LAMP reaction system.

[0027] The LAMP amplification reaction was incubated at 65 degrees Celsius for 120 minutes in a PCR instrument, and the fluorescence signal was detected every minute. Then, it was treated at 95 degrees Celsius for 2 minutes to inactivate the Bst 2.0 enzyme activity and terminate the reaction. The experiment was repeated 8 times. The results are shown in Figure 1 . Figure 1 The results showed that the primer sets of both genes underwent non-specific amplification (NSA) during the LAMP process. In particular, UL54 and UL75 showed non-specific amplification stably in all 8 experimental results, and the time points of the appearance of non-specific amplification products of UL54 and UL75 were 40.68 ± 2.078 minutes and 39.77 ± 0.721 minutes from the start of the reaction, respectively.

[0028] The above results show that the LAMP primer set used is the main factor causing false positive results in LAMP detection, and this influence is a common phenomenon. Example 2: LAMP assay using positive DNA template

[0029] Total DNA was extracted from human urine using a nucleic acid extraction kit, and the extracted total DNA was confirmed to contain cytomegalovirus DNA by conventional means, that is, it was a positive sample for human cytomegalovirus DNA. The number of copies of human cytomegalovirus DNA in the extracted sample was determined by fluorescent quantitative PCR, and then diluted so that the number of copies of human cytomegalovirus DNA added to the LAMP reaction system was 1000, 100, 10, and 1, respectively, and a primer control group without adding template DNA was set.

[0030] The primer sets targeting UL54 and UL75 in Example 1 were used to amplify the templates using the LAMP reaction system, and each copy number was repeated 8 times, and the average of the 8 repeated data was taken. The average results of the templates in this example at each copy number and the average results in Example 1 Figure 2 .

[0031] Figure 2 The results showed that within 60 minutes of reaction time, LAMP detected positive results for both positive and negative samples containing different numbers of viral DNA copies. Among them, the detection time of negative samples was even faster than the detection time of UL75 10 copies and 1 copy. Example 3: Adding blocking primers to inhibit non-specific amplification of LAMP

[0032] Design a blocking primer F1 that is complementary to the F1c portion of the upstream internal primer or a blocking primer B1 that is complementary to the B1c portion of the downstream internal primer. The schematic diagram of designing the blocking primer F1 or B1 is as follows: Figure 3As shown, 5' and 3' represent the 5' and 3' ends of the primers, respectively; 0 indicates that the blocking primer F1 (specifically named F1-SpC3) or B1 (specifically named B1-SpC3) is completely complementary to F1c or B1c; -3 indicates that the blocking primer F1 (specifically named F1-3C3P) or B1 (specifically named B1-3C3P) lacks 3 bases complementary to the 5' end F1c or 5' end B1c; -6 indicates that the blocking primer F1 (specifically named F1-3C6P) or B1 (specifically named B1-3C6P) lacks 6 bases complementary to the 5' end F1c or 5' end B1c; C3Spacer represents a spacer modification, which replaces the -OH on the 3rd carbon of the deoxyribose at the 3' end of the blocking primer with -OPO3H(CH2)3OH, so that the single-stranded blocking primer cannot form a 3-5 phosphodiester bond.

[0033] For the UL54 gene, the sequence of the blocking primer F1-SpC3-UL54 complementary to its upstream internal primer is shown in SEQ ID No. 13; the sequence of the blocking primer B1-SpC3-UL54 complementary to its downstream internal primer is shown in SEQ ID No.14.

[0034] For the UL75 gene, the sequence of the blocking primer F1-SpC3-UL75 complementary to its upstream internal primer is shown as SEQ ID No. 15; the sequence of the blocking primer B1-SpC3-UL75 complementary to its downstream internal primer is shown as SEQ ID No.16.

[0035] The LAMP reaction system was established based on the following primer sets: 1) Treatment group 1: FIP, F3, LF, F1-SpC3, B1-SpC3, BIP, B3 and LB; 2) Treatment group 2: FIP, F3, LF, F1-SpC3, BIP, B3 and LB; 3) Treatment group 3: FIP, F3, LF, B1-SpC3, BIP, B3 and LB; 4) Primer control group: FIP, F3, LF, BIP, B3 and LB.

[0036] The rest is the same as in Example 1.

[0037] A bar graph was drawn based on the time when the relative fluorescence unit in the amplification curve reached the baseline threshold, and each treatment group was subjected to one-way analysis of variance with the primer control group. The results are shown in Figure 4 .

[0038] Figure 4The experimental results showed that for the UL54 gene, the detection time of treatment group 1 was greater than 120 minutes, and no detection was found in treatment groups 2 or 3; for LU75, no detection was found in treatment groups 1 and 3, and the detection time of treatment group 2 was greater than 85 minutes. Usually, the detection time of LAMP is limited to within 90 minutes. The above results show that the use of blocking primers can completely or significantly inhibit the formation of non-specific products of the LAMP primer set. Example 4: Effect of blocking primers on LAMP positive amplification

[0039] The acquisition of human cytomegalovirus DNA was the same as in Example 2, and 1000 copies of human cytomegalovirus DNA were added to the reaction system of Example 3.

[0040] A bar graph was drawn based on the time when the relative fluorescence unit in the amplification curve reached the baseline threshold, and each treatment group was subjected to one-way analysis of variance with the primer control group. The results are shown in Figure 5 .

[0041] Figure 5 The experimental results showed that, for both the UL54 gene and the UL75 gene, the detection time of treatment groups 1 to 3 was prolonged compared with the primer control group, and the detection time of treatment group 1 was prolonged the most. Example 5: Effect of different lengths of blocking primers on nonspecific amplification

[0042] For the UL54 gene, the sequence of the blocking primer F1-3C3P-UL54 complementary to its upstream internal primer is shown as SEQ ID No. 17; the sequence of the blocking primer F1-3C6P-UL54 complementary to its upstream internal primer is shown as SEQ ID No. 18; the sequence of the blocking primer B1-3C3P-UL54 complementary to its downstream internal primer is shown as SEQ ID No. 19; and the sequence of the blocking primer B1-3C6P-UL54 complementary to its downstream internal primer is shown as SEQ ID No. 20.

[0043] For the UL75 gene, the sequence of the blocking primer F1-3C3P-UL75 complementary to its upstream internal primer is shown as SEQ ID No. 21; the sequence of the blocking primer F1-3C6P-UL75 complementary to its upstream internal primer is shown as SEQ ID No. 22; the sequence of the blocking primer B1-3C3P-UL75 complementary to its downstream internal primer is shown as SEQ ID No. 23; and the sequence of the blocking primer B1-3C6P-UL75 complementary to its downstream internal primer is shown as SEQ ID No. 24.

[0044] The LAMP reaction system was established based on the following primer sets: 1) Treatment group 1: FIP, F3, LF, F1-SpC3, B1-SpC3, BIP, B3, and LB; 2) Treatment group 2: FIP, F3, LF, F1-SpC3, BIP, B3, and LB; 3) Treatment group 3: FIP, F3, LF, B1-SpC3, BIP, B3, and LB; 4) Treatment group 4: FIP, F3, LF, F1-3C3P, B1-3C3P, BIP, B3, and LB; 5) Treatment group 5: FIP, F3, LF, F1-SpC3, BIP, B3, and LB. -3C3P, BIP, B3 and LB; 6) Treatment group 6: FIP, F3, LF, B1-3C3P, BIP, B3 and LB; 7) Treatment group 7: FIP, F3, LF, F1-3C6P, B1-3C6P, BIP, B3 and LB; 8) Treatment group 8: FIP, F3, LF, F1-3C6P, BIP, B3 and LB; 9) Treatment group 9: FIP, F3, LF, B1-3C6P, BIP, B3 and LB; 10) Primer control group: FIP, F3, LF, BIP, B3 and LB.

[0045] The rest is the same as in Example 1.

[0046] A bar graph was drawn based on the time when the relative fluorescence unit in the amplification curve reached the baseline threshold, and each treatment group was subjected to one-way analysis of variance with the primer control group. The results are shown in Figure 6 .

[0047] Figure 6 The experimental results show that for the target gene UL54, in the absence of viral template DNA, the use of blocking primers shortened by 3 bases or 6 bases can effectively avoid the occurrence of false positive results (no detection or detection time>120 minutes). Importantly, this inhibitory effect can be achieved even if any one blocking primer is used. For UL75, the use of full-length double blocking primers, a single full-length downstream internal primer blocking primer, a double blocking primer shortened by 3 bases, and a single downstream internal primer shortened by 3 bases can effectively avoid the occurrence of false positive results (no detection or detection time>120 minutes). The detection time of the blocking primers of a single full-length upstream internal primer, a single upstream internal primer shortened by 3 bases, a double blocking primer shortened by 6 bases, a single upstream internal primer shortened by 6 bases, and a single downstream internal primer shortened by 6 bases is greater than 75 minutes, about 80 to 85 minutes. Example 6: Effect of different blocking primer lengths on LAMP positive amplification sensitivity

[0048] The acquisition of human cytomegalovirus DNA was the same as in Example 2, and 1000 copies of human cytomegalovirus DNA were added to the reaction system of Example 5.

[0049] A bar graph was drawn based on the time when the relative fluorescence unit in the amplification curve reached the baseline threshold, and each treatment group was subjected to one-way analysis of variance with the primer control group. The results are shown in Figure 7 .

[0050] The experimental results are as follows Figure 7 As shown, the use of shortened blocking primers can significantly improve the sensitivity of LAMP reaction. Among them, for the UL54 gene, the average detection time of the primer control group is 10.44 minutes, the detection time using two full-length blocking primers is 38.33 minutes (full length), and the detection time using two blocking primers shortened by 3 bases is 18.61 minutes, and the detection time using two blocking primers shortened by 6 bases is 11.33 minutes. For the UL75 gene, the average detection time of the primer control group is 21.06 minutes, the detection time using two full-length blocking primers is 43.44 minutes, the detection time using two blocking primers shortened by 3 bases is 30.15 minutes, and the detection time using two blocking primers shortened by 6 bases is 19.89 minutes. It can be seen that the detection time is also controlled within 60 minutes. By combining this result with the result of Example 5, it can be seen that by controlling the inspection time within 60 minutes, the generation of false positives can be effectively avoided.

[0051] The above results show that the use of blocking primers can significantly reduce LAMP nonspecific amplification, and appropriately shortening the length of the blocking primers can shorten the detection time of positive amplification while significantly reducing LAMP nonspecific amplification, that is, appropriately shortening the length of the blocking primers can improve the sensitivity of LAMP amplification.

Claims

1. A blocking primer for reducing non-specific amplification of LAMP, which is completely complementary or partially complementary to the F1c fragment in the upstream internal primer and / or the B1c fragment in the downstream internal primer in the internal primer pair in the LAMP primer set, and has a C3 spacer modification at its 3' end.

2. The blocking primer according to claim 1, characterized in that When the blocking primer is partially complementary to the F1c fragment and / or the B1c fragment, the blocking primer lacks 3 to 6 bases complementary to the 5' end of the F1c fragment and / or the B1c fragment.

3. The blocking primer according to claim 1, characterized in that When the blocking primer is partially complementary to the F1c fragment and / or the B1c fragment, the blocking primer lacks 3 bases complementary to the 5' end of the F1c fragment and / or the B1c fragment.

4. The blocking primer according to claim 1, characterized in that For the UL54 gene of human cytomegalovirus, the sequence of the blocking primer is at least one of SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20; For the UL75 gene of human cytomegalovirus, the sequence of the blocking primer is at least one of SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No.

24.

5. A LAMP method for reducing non-specific amplification of LAMP, characterized in that: 1) Prepare a LAMP amplification reaction system: The LAMP amplification reaction system includes at least a LAMP primer set, a blocking primer according to any one of claims 1 to 3, a DNA sample for detection, dNTP, a LAMP fluorescent dye, and Mg. 2+ , an enzyme for LAMP amplification and a buffer for LAMP amplification; wherein the LAMP primer set consists of an internal primer pair, an external primer pair and a loop primer pair; 2) LAMP amplification reaction: Incubate at 65 degrees Celsius for 50 to 90 minutes, detect the fluorescence signal during the reaction, and then terminate the reaction.

6. The method according to claim 5, characterized in that In step 2), incubate at 65 degrees Celsius for 50 to 75 minutes.

7. The method according to claim 5, characterized in that In step 2), incubate at 65 degrees Celsius for 60 to 70 minutes.

8. The method according to claim 5, characterized in that In step 2), the activity of the enzyme used for LAMP amplification is inactivated at above 95 degrees Celsius to terminate the reaction.

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