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CN-121992082-A - Method and product for preventing false positive result in real-time fluorescence quantitative PCR detection pathogen

CN121992082ACN 121992082 ACN121992082 ACN 121992082ACN-121992082-A

Abstract

The present invention relates to methods and products for preventing false positive results in real-time fluorescent quantitative PCR detection of pathogens. The invention provides a positive reference template to replace a positive control template, thereby preventing false positive results caused by pollution of the positive control template to a test sample in the pathogen detection by real-time fluorescence quantitative PCR.

Inventors

  • YU JIE
  • PENG ZHENZHEN

Assignees

  • 南京诺唯赞动物保健有限公司
  • 南京诺唯赞生物科技股份有限公司

Dates

Publication Date
20260508
Application Date
20260209

Claims (10)

  1. 1. A polynucleotide comprising a reference region corresponding to a target region comprising at least a target upstream primer binding region and a target downstream primer binding region and a target probe binding region therebetween, the sequence of the target region being identical or reverse complementary to the sequence of a target segment of a target gene of a target species, the sequence of the target region being identical to or 3 ́ ends of the target upstream primer binding region comprising the sequence of the target upstream primer binding region, and the sequence of the target upstream primer and the sequence of the target downstream primer binding region being reverse complementary to the target downstream primer binding region or 3 ́ ends comprising the reverse complement of the target downstream primer binding region together are capable of amplifying the target region with the target region or a polynucleotide comprising the target region as a template, and a target probe having the same sequence as the target probe binding region or reverse complementary to the target region is capable of hybridizing to the target region or an amplified product thereof, the reference region being a contiguous sequence of the target region (e.g., a sequence of at least 35 to a contiguous sequence of the target region is replaced by a sequence of at least 3 nucleotides (e.g., at least 3 nucleotides at least 3 to 3 nucleotides or 3 to 35 nucleotides) in the target region).
  2. 2. A polynucleotide comprising an N-th reference region, N being a natural number of 2 or more, wherein the N-th reference region corresponds to an N-th target region comprising at least an N-th target upstream primer binding region and an N-th target downstream primer binding region and an N-th target probe binding region therebetween, the sequence of the N-th target region being identical to or reverse complementary to the sequence of an N-th target region of an N-th target gene of an N-th target species, the sequence of the N-th target region being identical to or 3 ́ end of the N-th target upstream primer binding region comprising the sequence of the N-th target upstream primer binding region and the N-th target downstream primer of which the sequence is reverse complementary to the N-th target downstream primer binding region or 3 ́ end of the N-th target downstream primer binding region comprising the reverse complementary sequence together being capable of amplifying the N-th target region or a polynucleotide comprising the N-th target region as a template, an nth target probe having the same or reverse complement sequence as the nth target probe binding region is capable of hybridizing to the nth target region or an amplification product thereof, the nth reference region being obtained by replacing the nth target upstream primer binding region in the nth target region with an nth reference upstream primer binding region having a different sequence (e.g., at least 3 ́ terminal 4 consecutive nucleotides are different) and/or replacing the nth target downstream primer binding region in the nth target region with an nth reference downstream primer binding region having a different sequence (e.g., at least 5 ́ terminal 4 consecutive nucleotides are different), N being a natural number of 1 to N.
  3. 3. A polynucleotide according to claim 2, wherein: (1) N target areas differing from each other or (2) At least two of the N target genes are different from each other, or (3) At least two of the N target species are different from each other.
  4. 4. A set of polynucleotides comprising N polynucleotides, N being a natural number of 2 or more, wherein the nth polynucleotide comprises an nth reference region corresponding to an nth target region comprising at least an nth target upstream primer binding region and an nth target downstream primer binding region and an nth target probe binding region therebetween, the sequence of the nth target region being identical to or reverse complementary to the sequence of an nth target segment of an nth target gene of an nth target species, the sequence of the nth target region being identical to or reverse complementary to the sequence of the nth target upstream primer binding region or the sequence of the nth target upstream primer binding region at the 3 ́ end thereof being complementary to the nth target downstream primer binding region or the sequence of the nth target downstream primer binding region at the 3 ́ end thereof being complementary to the nth target downstream primer binding region, together being capable of amplifying the nth target region or the polynucleotide comprising the nth target region as a template, an nth target probe having the same or reverse complement as the nth target probe binding region, capable of hybridizing to the nth target region or an amplification product thereof, the nth reference region being obtained by replacing the nth target upstream primer binding region in the nth target region with an nth reference upstream primer binding region having a different sequence (e.g., at least 3 ́ end 4 consecutive nucleotides are different) and/or replacing the nth target downstream primer binding region in the nth target region with an nth reference downstream primer binding region having a different sequence (e.g., at least 5 ́ end 4 consecutive nucleotides are different), n is a natural number from 1 to N.
  5. 5. The set of polynucleotides of claim 4, wherein: (1) N target areas differing from each other or (2) At least two of the N target genes are different from each other, or (3) At least two of the N target species are different from each other.
  6. 6. A method for determining the presence or absence of a target region in a test sample derived from a host species, comprising performing real-time fluorescent quantitative PCR on a test tube and a positive reference tube simultaneously, The test tube is provided with a test sample containing a test nucleic acid as a template, a target upstream primer and a target downstream primer as primer pairs, and a target probe as a probe, The positive reference tube is provided with a positive reference containing a positive reference polynucleotide as a template, a reference upstream primer and a reference downstream primer as primer pairs, and a target probe as a probe, Said positive reference polynucleotide comprising a reference region corresponding to a target region comprising at least a target upstream primer binding region and a target downstream primer binding region and a target probe binding region therebetween, said target region having a sequence identical to or reverse complementary to a sequence of a target segment of a target gene of a target species, said target upstream primer binding region or said target upstream primer comprising a sequence of said target upstream primer binding region at its 3 ́ end and said target downstream primer binding region being reverse complementary to said target downstream primer binding region or said target downstream primer comprising a reverse complementary sequence of said target downstream primer binding region at its 3 ́ end together being capable of amplifying said target region using said target region or a polynucleotide comprising said target region as a template, said target probe having a sequence identical to or reverse complementary to said target probe binding region being capable of hybridizing to said target region or an amplification product thereof, said reference region being obtained by sequential substitution of at least 3 nucleotide(s) of said target upstream primer binding region in said target region with at least 3 nucleotide(s) or at least 3 nucleotide(s) of said target region (e.g., at least 35 to a contiguous nucleotide(s) of said target region of interest) in sequence (e.g., at least 35 to a contiguous nucleotide (e.g., 35) of said target region), the reference upstream primer having the same sequence as the reference upstream primer binding region or comprising the sequence of the reference upstream primer binding region at its 3 ́ terminus and the reference downstream primer having the sequence reverse complementary to the reference downstream primer binding region or comprising the reverse complement of the reference downstream primer binding region at its 3 ́ terminus are together capable of amplifying the reference region using the reference region or a polynucleotide comprising the reference region as a template.
  7. 7. A method for determining the presence or absence of N target regions in a test sample derived from a host species, N being a natural number greater than 2, comprising performing real-time fluorescent quantitative PCR on N test tubes and N positive reference tubes simultaneously, An nth test tube is provided with a test sample containing a test nucleic acid as a template, an nth target upstream primer and an nth target downstream primer as primer pairs, and an nth target probe as a probe, An nth positive reference tube is provided with a positive reference containing a positive reference polynucleotide as a template, an nth reference upstream primer and an nth reference downstream primer as primer pairs, and an nth target probe as a probe, The positive reference polynucleotide comprises an nth reference region corresponding to an nth target region comprising at least an nth target upstream primer binding region and an nth target downstream primer binding region with an nth target probe binding region therebetween, the sequence of the nth target region being identical to or reverse complementary to the sequence of an nth target segment of an nth target gene of an nth target species, the sequence of the nth target upstream primer binding region being identical to or 3 ́ end of the nth target upstream primer binding region comprising the sequence of the nth target upstream primer binding region and the sequence of the nth target upstream primer binding region being reverse complementary to the nth target downstream primer binding region or 3 ́ end of the nth target downstream primer binding region comprising the reverse complementary sequence of the nth target downstream primer binding region together being capable of amplifying the nth target region using the nth target region or a polynucleotide comprising the nth target region as a template, said nth target probe having the same or reverse complement as said nth target probe binding region being capable of hybridizing to said nth target region or an amplification product thereof, said nth reference region being obtained by replacing said nth target upstream primer binding region in said nth target region with an nth reference upstream primer binding region having a different sequence (e.g., having at least 3 ́ ends and 4 consecutive nucleotides being different) and/or replacing said nth target downstream primer binding region in said nth target region with an nth reference downstream primer binding region having a different sequence (e.g., having at least 5 ́ ends and 4 consecutive nucleotides being different), the nth reference upstream primer having the same sequence as the nth reference upstream primer binding region or comprising the sequence of the nth reference upstream primer binding region at the 3 ́ terminus and the nth reference downstream primer having the sequence reverse complementary to the nth reference downstream primer binding region or comprising the reverse complement of the nth reference downstream primer binding region at the 3 ́ terminus together are capable of amplifying the nth reference region with the nth reference region or a polynucleotide comprising the nth reference region as a template, N being a natural number of 1 to N.
  8. 8. The method of claim 7, wherein: (1) The N positive reference polynucleotides comprise N reference regions, the N positive reference polynucleotides comprise N reference regions, or (2) The N reference regions are contained in a single positive reference polynucleotide.
  9. 9. The method of claim 7, wherein: (1) N target areas differing from each other or (2) At least two of the N target genes are different from each other, or (3) At least two of the N target species are different from each other.
  10. 10. A kit, comprising: (1) The polynucleotide of claim 1; (2) A polynucleotide according to claim 2 or 3, or (3) The set of polynucleotides of claim 4 or 5.

Description

Method and product for preventing false positive result in real-time fluorescence quantitative PCR detection pathogen Technical Field The present invention relates to methods and products for preventing false positive results in real-time fluorescent quantitative PCR detection of pathogens. The invention provides a positive reference template to replace a positive control template, thereby preventing false positive results caused by pollution of the positive control template to a test sample in the pathogen detection by real-time fluorescence quantitative PCR. Background The technology for detecting pathogens by real-time fluorescence quantitative PCR has been widely used because of the outstanding advantages of high sensitivity, strong specificity, rapid diagnosis, simple operation, good repeatability, high automation degree, easy standardized operation, high biological safety of test and the like, but the pollution source is strictly controlled because of the fact that very little pollution is likely to cause false positive of detection results due to the high sensitivity. The positive control in the commercial qPCR diagnostic kit is one of pollution sources, and becomes a difficult problem for experimenters to judge the negative and positive of the sample. Patent CN106868202a discloses a method for monitoring pollution of fluorescent quantitative PCR reaction, which uses an external reference gene as an additional positive reference and a primer probe matched with the external reference gene to be added into a reaction system so as to achieve the purpose of monitoring whether a sample to be detected is polluted by a positive control in the experimental process. However, because the reference gene positive reference and the target positive control are independent plasmids, the risk of misjudgment still exists in the pollution monitoring process of the positive control of the kit, and the interference on the target detection reaction can be increased by adding a pair of primer probes in the reaction system. The patent CN116334314A provides a fluorescence PCR positive control for distinguishing the African swine fever virus of a sample to be tested polluted by a positive control, wherein the positive control is a positive control subjected to sequence modification, a part of the region of a target sequence of the P72 gene of the African swine fever virus amplified by a specific detection primer is selected to be changed into an internal standard probe sequence, but no means is provided for eliminating a false positive result caused by the positive control pollution in a sequence modification mode. Disclosure of Invention The invention aims to provide a method for solving the problem of false positive caused by positive control aerosol pollution. The method has the beneficial effects that according to experimental data, the false positive result caused by the positive control aerosol pollution can be eliminated on the basis that the sensitivity and the specificity of the qPCR diagnosis kit are not affected, the positive control can be stably stored, and the method meets the requirements of commercial kits. In one aspect, the invention provides polynucleotides that serve as positive reference templates, replacing conventional positive control templates. The positive reference template can prevent false positive results caused by pollution of a positive control to a test sample in the pathogen detection by real-time fluorescence quantitative PCR. That is, even a true negative test sample contaminated with the positive reference template of the present invention does not produce a false positive result due to the contaminated positive reference template of the present invention when detecting a pathogen by the real-time fluorescent quantitative PCR of the present invention. In a first aspect, the invention provides a polynucleotide comprising a reference region, said reference region corresponding to a target region, said target region comprising at least a target upstream primer binding region and a target downstream primer binding region and a target probe binding region therebetween, said target region having a sequence identical to or complementary to the sequence of a target region of a target gene of a target species, said target region having a sequence identical to or complementary to the sequence of said target upstream primer binding region at the 3 ́ end comprising the sequence of said target upstream primer binding region, and a target upstream primer having a sequence complementary to the sequence of said target downstream primer binding region or complementary to the sequence of said target downstream primer binding region at the 3 ́ end comprising the reverse complement of said target downstream primer binding region, together being capable of amplifying said target region using said target region or a polynucleotide comprising said target region as a template, said target region having a sequenc