AU2020100696A4 - LAMP-TaqMan ASSAY KIT FOR PIGEON NEWCASTLE DISEASE VIRUS - Google Patents
LAMP-TaqMan ASSAY KIT FOR PIGEON NEWCASTLE DISEASE VIRUS Download PDFInfo
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Abstract
The present invention provides an LAMP-TaqMan assay kit for pigeon Newcastle disease virus (NDV). The advantages of loop-mediated isothermal amplification (LAMP) lie in high specificity and sensibility, simplicity, convenience, and low cost. In the present invention, using NDV F gene as a target gene, and integrating these advantages of LAMP with a TaqMan probe, an LAMP-TaqMan assay for pigeon NDV is established and an LAMP-TaqMan assay kit for pigeon NDV is constructed based on the assay. The assay kit of the present invention includes six specific primers and a TaqMan fluorescent probe, and nucleotide sequences thereof are shown in SEQ ID NO. 1 to 7, respectively. The kit of the present invention detects 10 copies/ l standard plasmids effectively, but reaction time is merely 25 min. Results demonstrate that the kit of the present invention achieves rapid and accurate detection of the pigeon NDV, with a good application prospect.
Description
LAMP-TaqMan ASSAY KIT FOR PIGEON NEWCASTLE DISEASE VIRUS TECHNICAL FIELD
The present invention relates to the technical field of PCR detection, and in particular to an LAMP-TaqMan assay kit for pigeon Newcastle disease virus.
BACKGROUND
Newcastle disease (ND), also known as paramyxovirosis, is an acute infectious disease caused by pigeon paramyxovirus Type 1 (PPMV-1), a variant of Newcastle disease virus (NDV). The disease is one of the main diseases endangering the pigeon industry, causing great economic losses for the pigeon industry. So far, ND is mainly detected through virus isolation and culture, 10 hemagglutination assay (HA), hemagglutination inhibition (HI) test, fluorescent antibody test, and conventional PCR. These detection methods not only asks for high requirements for instrument and laboratory environment, but also features complex operation, strict technical requirements of operators, and long detection time.
Therefore, it is urgent to establish a rapid, simple and highly specific technique of pigeon NDV 15 detection, so as to meet the detection requirement. The priority is to establish a rapid screening assay with high sensitivity and accuracy.
Loop-mediated isothermal amplification (LAMP) is an emerging gene amplification technique developed by Japanese scholar Tsugunori Notomi in 2000, featuring strong specificity, high sensitivity, isothermal efficiency, easy operation, and low cost. However, LAMP cannot amplify 20 long strands of DNA, because this is strand displacement amplification, and the length of target sequence is within 300 bp, but a length of >500 bp enables difficult amplification. Because of high sensitivity, false positive results may result from contamination easily. Therefore, special attention should be paid to rigorous operation for fear of nonspecific amplification and contamination. SUMMARY
According to one aspect of the present invention there is provided a LAMP-TaqMan assay kit for pigeon Newcastle disease virus (NDV) and an assay based on LAMP and TaqMan assay.
The assay kit of the present invention includes six specific primers and a TaqMan fluorescent probe, the kit uses NDV F gene as a target gene, and a nucleotide sequence of the target gene is shown in SEQ ID NO. 8; the six specific primers include two outer primers, two inner primers, and 30 two loop primers, respectively.
Preferably, the two outer primers are:
NVDF2 F3: 5’-TCATTAATAGGCAGTGGCT-3’
NVDF2 B3: 5’-TGTACAGTATAGATCCtGATC-3’;
the two inner primers are:
NVDF2 FIP: 5’-AGGTTCCCGACTGAGGGTACTGTATGACTCACAGACTC-3’
NVDF2 BIP: 5’-TTGCCTCAGCACTTGTCCGGTGTCAAGTTCTTCTATCAC-3’;
2020100696 04 May 2020 the two loop primers are:
NVDF2 LoopF: 5’-AATTTACCTGGATGCCCAAG-3’
NVDF2 LoopB: 5’-CGAAGGTAGTGACACAAGTC-3’.
Preferably, the TaqMan probe is:
5’-GTGCCACCTACCTGGAAACTTTATCTGTAAGCACAA -3’.
When the kit of the present invention is used, concentrations of the primers and the probe may be as follows: primers: NVDF2 F3: 2 μΜ, NVDF2 B3: 2 μΜ; NVDF2 LoopF: 8 pM, NVDF2 LoopB: 8 pM; NVDF2 FIP: 16 pM, NVDF2 BIP: 16 pM; fluorescent probe: 8 pM.
The kit of the present invention can further include dNTP, Bst5.0 polymerase, trehalose, 10 negative and positive controls. The positive control is positive plasmids of pigeon NDV.
According to another aspect of the present invention there is provided a primer/probe combination for detecting pigeon NDV, including six specific primers and a TaqMan probe. The six specific primers include two outer primers, two inner primers, and two loop primers, respectively; nucleotide sequences thereof are shown in SEQ ID NO. 1 to 6, respectively; the nucleotide 15 sequence of the TaqMan probe is shown in SEQ ID NO. 7; a fluorescence reporter and a fluorescence quencher are labeled on either end of the probe.
In examples of the present invention, the probe is: 5’-(FAM) GTGCCACCTACCTGGAAACTTTATCTGTAAGCACAA (BHQ)-3 ’.
According to another aspect of the present invention there is provided an LAMP-TaqMan assay 20 for pigeon Newcastle disease virus, where, a genomic DNA of an analyte is used as a template, the LAMP-TaqMan assay is conducted on the template by the primer/probe combination, and fluorescence signals of FAM are collected by a thermostatic fluorescence analyzer or a standard quantitative PCR system; the analyte with an ascending curve is determined as positive, while that without ascending curve is determined as negative.
In the foregoing assay, a detection system of the LAMP-TaqMan assay can include: primers: NVDF2 F3: 2 μΜ, NVDF2 B3: 2 μΜ; NVDF2 LoopF: 8 μΜ, NVDF2 LoopB: 8 μΜ; NVDF2 FIP: 16 μΜ, NVDF2 BIP: 16 μΜ; fluorescent probe: 8 μΜ.
A procedure of the LAMP-TaqMan assay is: specific amplification at 65°C for 10 s; extension at 65°C for 50 s to collect fluorescence signals, for 25 cycles, with a total reaction time of 25 min.
Preferred embodiments of the present invention can have the following benefits. The advantages of LAMP lie in high specificity and sensibility, simplicity, convenience, and low cost. In preferred embodiments of the present invention, using NDV F gene as a target gene, and integrating these advantages of LAMP with a TaqMan probe, an LAMP-TaqMan assay for pigeon NDV is established and an LAMP-TaqMan assay kit for pigeon NDV is constructed based on the assay. The kit can effectively detects 10 copies/μΐ standard plasmids, but reaction time is merely 25 min. Results demonstrate that the kit of the present invention can achieve rapid and accurate detection of
2020100696 04 May 2020 the pigeon NDV, with a good application prospect.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 illustrates results of detection of different RNA consumptions with the kit of a preferred embodiment of the present invention after extraction of RNAs from virus-infected tissue Al. 5 Consumption: 10 ng, 1 ng, 0.1 ng, 0.01 ng, 1 pg, 0.1 pg, 0.01 pg, and NTC. Amounts of RNA templates added in a 25 μΐ LAMP-TaqMan reaction system are as follows: 1: 10 ng; 2: 1 ng; 3: 0.1 ng; 4: 0.01 ng; 5: 1 pg; 6: 0.1 pg; 7: 0.01 pg; and 8: ddELO (NTC). Using the method, 0.1 ng of extracted RNA molecules can be detected within 25 min, but less than 0.01 ng of sample cannot be detected, without fluorescence signal.
FIG. 2 illustrates results of sensitivity testing for standard plasmid templates using the kit of a preferred embodiment of the present invention. Concentrations of templates for amplification added in a 25 μΐ LAMP-TaqMan reaction system are as follows: 1: 107 copies/μΐ; 2: 106 copies/μΐ; 3: 105 copies/μΐ; 4: 104 copies/μΐ; 5: 103 copies/μΐ; 6: 102 copies/μΐ; 7: 10 copies/μΐ; and 8: 0 copy/μΐ (NTC). Using the method, virus detection can be completed within 25 min, and negative 15 amplification shows no fluorescence signal.
DETAILED DESCRIPTION
The following examples are intended to further illustrate the protection content of the present invention but not to limit the protection scope of the present invention. Modifications or substitutions made to methods, steps or conditions of the present invention are deemed to fall within 20 the scope of the present invention, without departing from the spirit and essence of the present invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, all biochemical reagents and materials in examples of the present application are commercially available.
Example 1 Determination of primers/probe of LAMP-TaqMan assay for pigeon Newcastle disease virus (NDV)
Focusing on a fragment of a highly conserved domain of pigeon NDV F gene (SEQ ID NO. 8), the present inventor designed a combination of six primers using LAMP Designer software according to the principle of LAMP primer design. The principle was to ensure an interval of more 30 than 50 bp between 3’-terminal of Fl primer and 5’-terminal of Bl primer, so as to favor the design of a TaqMan probe. After completion of the LAMP primer design, the TaqMan probe was designed based on the LAMP primer. Design principles of the TaqMan probe were as follows: the length was 30 to 45 bp; TM was above 72°C; the probe of the LAMP primer was designed 4 to 8 bp away from the 3’-terminal of Fl primer. Primers were analyzed by DNAMAN, and a few candidate primers 35 and probes were selected.
A total of three primer/probe combinations were screened out by the kit. Sequences are listed in
2020100696 04 May 2020
Table 1.
Table 1 Three primer/probe combinations designed based on a fragment of a highly conserved domain of pigeon NDV F gene
| 3NVDF F3 | TCATTAATAGGCAGTGGCT | Combination 1 is the optimal primer/probe combination |
| 3NVDF B3 | TGTACAGTATAGATCCtGATC | |
| 3NVDF F1P | AGGTTCCCGACTGAGGGTACTGTATGACTCACAGACTC | |
| 3NVDF BIP | TTGCCTCAGCACTTGTCCGGTGTCAAGTTCTTCTATCAC | |
| 3NVDF LoopF | AATTTACCTGGATGCCCAAG | |
| 3NVDF LoopB | CGAAGGTAGTGACACAAGTC | |
| 3NVDF Probe | GTGCCACCTACCTGGAAACTTTATCTGTAAGCACAA | |
| 2NVDF F3 | CAACCTACCTGGAGACTTT | Combination 2 is eliminated |
| 2NVDF B3 | CAATGACTGAGCCTTTGAG | |
| 2NVDF FIP | ACAGTATGAGGTGTCGAGTTCTCAAAGGATTTGCCTCAGC | |
| 2NVDF BIP | ATTCCCCATGTCTCCAGGTATTGCCTTCAGTCTTTGAATA CAT | |
| 2NVDF LoopF | AGCCGACTTGTGTCACTA | |
| 2NVDF LoopB | ATTCTTGTCTGAGCGGTAATAC | |
| 2NVDF Probe | ATCTGATCTGGATCTATACTGTACAAAGATAGT | |
| 1NVDF F3 | TTAATAATATGCGTGCCACC | Combination 3 is eliminated |
| 1NVDF B3 | AATGACTGAGCCCTTGAG | |
| 1NVDF FIP | TCTATCACAGAGCCGACTTGTGTAAGCACAACCAAAGGG | |
| 1NVDF BIP | ACATTCCCTATGTCTCCAGGAACCTTCAGTCTTTGAGTACA TG | |
| 1NVDF LoopF | GTCACTACCTTCGGGACA | |
| 1NVDF LoopB | ATTCTTGTCTGAGCGGTAATAC | |
| 1NVDF Probe | TAGAATCTGATCTGGATCGATACTGTACAAAGATAGT |
Combination 1 was highly specific and sensitive, while Combinations 2 and 3 produced dimers.
Therefore, Combinations 2 and 3 were eliminated, and Combination 1 was selected as the primers of the kit.
Finally, the example determined six specific primers and a TaqMan probe in the assay kit for pigeon NDV as the following combination, focusing on nine binding domains.
The two outer primers were:
2020100696 04 May 2020
NVDF2 F3: 5’-TCATTAATAGGCAGTGGCT-3’ (SEQ ID NO. 1)
NVDF2 B3: 5’-TGTACAGTATAGATCCtGATC-3’; (SEQ ID NO. 2) the two inner primers were:
NVDF2 FIP: 5’-AGGTTCCCGACTGAGGGTACTGTATGACTCACAGACTC-3’ (SEQ ID
NO. 3)
NVDF2 BIP: 5’-TTGCCTCAGCACTTGTCCGGTGTCAAGTTCTTCTATCAC-3’ (SEQ ID NO. 4);
the two loop primers were:
NVDF2 LoopF: 5’-AATTTACCTGGATGCCCAAG-3’ (SEQ ID NO. 5)
NVDF2 LoopB: 5’-CGAAGGTAGTGACACAAGTC-3’ (SEQ ID NO. 6).
The TaqMan probe was: 5’-(FAM) GTGCCACCTACCTGGAAACTTTATCTGTAAGCACAA (BHQ)-3’(SEQ ID NO. 7).
Example 2 Establishment of LAMP-TaqMan assay for pigeon Newcastle disease virus (NDV)
LAMP-TaqMan assay was conducted on positive plasmids of pigeon NDV using the primer/primer combination determined in Example 1.
Detection system was as follows:
The procedure of the assay was: specific amplification at 65°C for 10 s; extension at 65°C for 50 s to collect fluorescence signals, for 25 cycles, with a total reaction time of 25 min.
A negative NTC sample showed no peak, while experimental conditions were satisfied under the condition that a positive standard (NDVF-pJET plasmid was amplified by RT-PCR and conjugated to a pJET-T vector; the standard plasmid refers to the following reference: [LI FH, et al. Isolation and identification of pigeon Newcastle disease virus and experimental infection in pigeons. Progress in Veterinary Medicine, 2016, 37(9):57-61]) showed a peak. In case of a peak, an analyte was labeled as containing NDV RNA molecules. After extraction of RNAs from virus-infected tissue Al, different RNA consumptions were tested by the assay established in preferred embodiments of the present invention or by the kit constructed based on the specific primer/probe combination designed in preferred embodiments of the present invention. Results are illustrated in FIG. 1. It was indicated that 0.1 ng of extracted RNA molecules could be detected by the assay and the kit within 25 min, but less than 0.01 ng of sample could not be detected, without fluorescence signal. Therefore, pigeon NDV can be tested specifically and sensitively.
Example 3 Sensitivity testing
Positive plasmids of pigeon NDV were diluted 1:10. There were seven dilutions, ranging from 10® to 10’7. LAMP-TaqMan amplification was conducted according to the assay established in 35 Example 2 in order to determine the sensitivity of real-time fluorescent LAMP assay established by our institute.
2020100696 04 May 2020
Sensitivity testing results indicated that, using NDVF-pJET plasmid as test template, LAMP-TaqMan assay detected 10 copies/μΐ standard plasmids effectively, but reaction time was merely 25 min. Results are illustrated in FIG. 2.
Although the present invention has been described in detail above with a general description 5 and specific examples, some modifications or improvements can be made on the basis of the present invention, which is apparent to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the protection scope of the present invention.
Throughout this specification and the claims which follow, unless the context requires 10 otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Claims (5)
1. An LAMP-TaqMan assay kit for pigeon Newcastle disease virus (NDV), wherein the kit comprises six specific primers and a TaqMan fluorescent probe, the kit uses NDV F gene as a target gene, and a nucleotide sequence of the target gene is shown in SEQ ID NO. 8; the six specific primers comprise two outer primers, two inner primers, and two loop primers, respectively.
2. The assay kit according to claim 1, wherein the two outer primers are:
NVDF2 F3: 5’-TCATTAATAGGCAGTGGCT-3’
NVDF2 B3: 5’-TGTACAGTATAGATCCtGATC-3’;
the two inner primers are:
NVDF2 FIP: 5’-AGGTTCCCGACTGAGGGTACTGTATGACTCACAGACTC-3’
NVDF2 BIP: 5’-TTGCCTCAGCACTTGTCCGGTGTCAAGTTCTTCTATCAC-3’;
the two loop primers are:
NVDF2 LoopF: 5’-AATTTACCTGGATGCCCAAG-3’
NVDF2 LoopB: 5’-CGAAGGTAGTGACACAAGTC-3’;
wherein the TaqMan probe is:
5’- GTGCCACCTACCTGGAAACTTTATCTGTAAGCACAA -3’;
preferably, wherein concentrations of the primers and the probe are as follows: primers: NVDF2 F3: 2 μΜ, NVDF2 B3: 2 μΜ; NVDF2 LoopF: 8 μΜ, NVDF2 LoopB: 8 μΜ; NVDF2 FIP: 16 μΜ, NVDF2 BIP: 16 μΜ; fluorescent probe: 8 μΜ.
3. A primer/probe combination for detecting pigeon Newcastle disease virus (NDV), comprising six specific primers and a TaqMan probe, wherein the six specific primers comprise two outer primers, two inner primers, and two loop primers, respectively; nucleotide sequences thereof are shown in SEQ ID NO. 1 to 6, respectively; the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 7; a fluorescence reporter and a fluorescence quencher are labeled on either end of the probe.
4. Use of the primer/probe combination according to claim 3 in preparation of an assay kit for pigeon Newcastle disease virus.
5. An LAMP-TaqMan assay for pigeon Newcastle disease virus, wherein, a genomic DNA of an analyte is used as a template, LAMP-TaqMan assay is conducted on the template by the primer/probe combination according to claim 6, and fluorescence signals of FAM are collected by a thermostatic fluorescence analyzer or a standard quantitative PCR system; the analyte with an ascending curve is determined as positive, while that without ascending curve is determined as negative;
preferably, wherein a detection system of the LAMP-TaqMan assay comprises: primers:
2020100696 20 May 2020
NVDF2 F3: 2 μΜ, NVDF2 B3: 2 μΜ; NVDF2 LoopF: 8 μΜ, NVDF2 LoopB: 8 μΜ; NVDF2 FIP: 16 μΜ, NVDF2 BIP: 16 μΜ; fluorescent probe: 8 μΜ;
preferably, wherein a procedure of the LAMP-TaqMan assay is: specific amplification at 65°C for 10 s; extension at 65°C for 50 s to collect fluorescence signals, for 25 cycles, with a total reaction time of 25 min.
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| CN201910753231.2 | 2019-08-15 | ||
| CN201910753231.2A CN110305988A (en) | 2019-08-15 | 2019-08-15 | Pigeon with newcastle disease LAMP-TaqMan detection kit |
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| CN110578019B (en) * | 2019-10-30 | 2023-04-28 | 广西壮族自治区兽医研究所 | Double-fluorescence LAMP (loop-mediated isothermal amplification) detection kit for distinguishing strong and weak viruses of newcastle disease and primer group thereof |
| CN111560483B (en) * | 2020-07-13 | 2020-11-03 | 元码基因科技（北京）股份有限公司 | Reaction system for detecting low-abundance novel coronavirus, method and application |
| CN113201584B (en) * | 2021-06-09 | 2022-11-04 | 湖南融健生物科技有限公司 | Detection method, kit and application of multiple target nucleic acids |
| CN114717231A (en) * | 2022-04-11 | 2022-07-08 | 江苏省农业科学院 | RT-PCR primer for detecting pigeon paramyxovirus, application thereof and RT-PCR kit containing primer |
| CN114807451A (en) * | 2022-06-08 | 2022-07-29 | 河北三狮生物科技有限公司 | Primer and probe for specifically detecting carp edema virus and application of primer and probe |
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| CN103540660B (en) * | 2013-10-10 | 2015-10-28 | 中国人民解放军疾病预防控制所 | Based on the loop-mediated isothermal amplification method of TaqMan probe and special LAMP primer thereof and test kit |
| CN108486279A (en) * | 2017-09-21 | 2018-09-04 | 山东省农业科学院家禽研究所 | A method of pigeon with newcastle disease is detected based on fluorescent quantitative PCR technique |
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