Method and product for preventing false positive result in real-time fluorescent quantitative PCR (polymerase chain reaction) detection of pathogens

Abstract

The invention relates to a method and a product for preventing false positive results in real-time fluorescent quantitative PCR detection of pathogens. The positive reference template provided by the invention is used for replacing a positive control template, so that a false positive result caused by the fact that a test sample is polluted by the positive control template in real-time fluorescent quantitative PCR detection of pathogens is prevented.

Description

Technical Field

[0001] This invention relates to methods and products for preventing false positive results in real-time quantitative PCR detection of pathogens. The invention provides a positive reference template as an alternative to a positive control template, thereby preventing false positive results in real-time quantitative PCR detection of pathogens caused by contamination of the test sample by the positive control template. Background Technology

[0002] Real-time quantitative PCR (qPCR) for pathogen detection has been widely used due to its outstanding advantages, including high sensitivity, high specificity, rapid diagnosis, simple operation, good repeatability, high degree of automation, easy standardization, and high biosafety. However, precisely because of its high sensitivity, even trace amounts of contamination can lead to false positives, making the control of contamination sources extremely strict. Positive controls in commercially available qPCR diagnostic kits are one source of contamination, posing a significant challenge for researchers in determining the positive or negative status of samples.

[0003] Patent CN106868202A discloses a method for monitoring contamination in quantitative real-time PCR reactions. This method uses an external reference gene as an additional positive control, along with its matching primers and probes, added to the reaction system to monitor whether the sample to be tested is contaminated by the positive control during the experiment. However, since the external reference gene and the target positive control are independent plasmids, there is still a risk of misjudgment in the monitoring of contamination of the positive control in the kit. Furthermore, adding an extra pair of primers and probes to the reaction system increases interference with the target detection reaction. Patent CN116334314A provides a positive control for African swine fever virus fluorescent PCR to distinguish positive control contamination of the sample to be tested. This positive control is a sequence-modified positive control, in which a portion of the target sequence of the African swine fever virus P72 gene amplified by the specific detection primers is modified into an internal standard probe sequence. However, no method has been found to eliminate false positive results caused by positive control contamination through sequence modification. Summary of the Invention

[0004] The purpose of this invention is to provide a method to solve the problem of false positives caused by aerosol contamination of positive control.

[0005] Beneficial effects: According to experimental data, the method of the present invention can eliminate false positive results caused by aerosol contamination of the positive control without affecting the sensitivity and specificity of the qPCR diagnostic kit, and the positive control can be stably preserved, which meets the requirements of commercial kits.

[0006] On the one hand, this invention provides a polynucleotide that serves as a positive reference template, replacing conventional positive control templates. The positive reference template of this invention can prevent false positive results in real-time quantitative PCR detection of pathogens caused by contamination of test samples with positive controls. In other words, even true negative test samples contaminated with the positive reference template of this invention will not produce false positive results when using real-time quantitative PCR to detect pathogens due to contamination with the positive reference template of this invention.

[0007] In a first aspect, the present invention provides a polynucleotide comprising a reference region corresponding to a target region, the target region comprising at least a target upstream primer binding region and a target downstream primer binding region, and a target probe binding region between them, wherein the sequence of the target region is identical to or inversely complementary to the sequence of a target segment of a target gene of a target species, a target upstream primer whose sequence is identical to or whose 3' end contains the sequence of the target upstream primer binding region, and a target downstream primer whose sequence is inversely complementary to or whose 3' end contains the inversely complementary sequence of the target downstream primer binding region, together, can amplify the target region using the target region or a polynucleotide containing the target region as a template, and a target probe whose sequence is identical to or inversely complementary to the target probe binding region can hybridize with the target region or its amplification product, wherein the reference region is obtained by using the target... The target upstream primer binding region in the region is obtained by replacing it with a reference upstream primer binding region with a different sequence (e.g., at least 4, 6, or 8 consecutive nucleotides different at the 3' end, such as 10-50 consecutive nucleotides, 15-40 consecutive nucleotides, 20-30 consecutive nucleotides, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides different) and / or by replacing the target downstream primer binding region in the target region with a reference downstream primer binding region with a different sequence (e.g., at least 4, 6, or 8 consecutive nucleotides different at the 5' end, such as 10-50 consecutive nucleotides, 15-40 consecutive nucleotides, 20-30 consecutive nucleotides, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides different).

[0008] In a second aspect, the present invention provides a polynucleotide comprising N reference regions, where N is a natural number greater than or equal to 2, wherein the nth reference region corresponds to the nth target region, and the nth target region comprises at least an nth target upstream primer binding region and an nth target downstream primer binding region, as well as an nth target probe binding region between them. The sequence of the nth target region is identical to or reverse complementary to the sequence of the nth target segment of the nth target gene of the nth target species. An nth target upstream primer whose sequence is identical to or whose 3' end contains the sequence of the nth target upstream primer binding region, and an nth target downstream primer whose sequence is reverse complementary to or whose 3' end contains the reverse complementary sequence of the nth target downstream primer binding region, together can amplify the nth target region using the nth target region or a polynucleotide containing the nth target region as a template. An nth target probe whose sequence is identical to or reverse complementary to the nth target probe binding region can bind to the nth target region or its amplified product. In the hybridization process, the nth reference region is formed by replacing the nth target upstream primer binding region in the nth target region with a nth reference upstream primer binding region that has a different sequence (e.g., at least 4, 6, or 8 consecutive nucleotides different at the 3' end, e.g., 10-50 consecutive nucleotides, 15-40 consecutive nucleotides, 20-30 consecutive nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides different). / Or, the nth target downstream primer binding region in the nth target region can be obtained by replacing it with a reference downstream primer binding region with a different sequence (e.g., at least 4, at least 6, or at least 8 consecutive nucleotides different at the 5' end, e.g., 10-50 consecutive nucleotides, e.g., 15-40 consecutive nucleotides, e.g., 20-30 consecutive nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides different), where n is a natural number from 1 to N.

[0009] In one implementation, the N target regions are pairwise distinct. In one implementation, at least two of the N target genes are distinct from each other. In one implementation, at least two of the N target species are distinct from each other.

[0010] In one implementation, two or more target regions target the same target gene. In another implementation, two or more target regions target the same target species.

[0011] In a third aspect, the present invention provides a set of polynucleotides comprising N polynucleotides, where N is a natural number greater than 2. The nth polynucleotide contains an nth reference region, which corresponds to the nth target region. The nth target region contains at least an nth target upstream primer binding region and an nth target downstream primer binding region, as well as an nth target probe binding region between them. The sequence of the nth target region is identical to or reverse complementary to the sequence of the nth target segment of the nth target gene of the nth target species. An nth target upstream primer whose sequence is identical to or whose 3' end contains the sequence of the nth target upstream primer binding region, and an nth target downstream primer whose sequence is reverse complementary to or whose 3' end contains the reverse complementary sequence of the nth target downstream primer binding region, together with the nth target region or a polynucleotide containing the nth target region, can amplify the nth target region using the nth target region or a polynucleotide containing the nth target region as a template. An nth target probe whose sequence is identical to or reverse complementary to the nth target probe binding region can bind to the nth target target region. The nth target region or its amplification product is hybridized, wherein the nth reference region is obtained by binding the nth target upstream primer binding region in the nth target region with a nth reference upstream primer whose sequence is different (e.g., at least 4, at least 6, or at least 8 consecutive nucleotides different at the 3' end, for example, 10-50 consecutive nucleotides, for example, 15-40 consecutive nucleotides, for example, 20-30 consecutive nucleotides, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides different). The nth target downstream primer binding region is obtained by replacing the nth target region with a reference downstream primer binding region that has a different sequence (e.g., at least 4, 6, or 8 consecutive nucleotides different at the 5' end, e.g., 10-50 consecutive nucleotides, e.g., 15-40 consecutive nucleotides, e.g., 20-30 consecutive nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides different), where n is a natural number from 1 to N.

[0012] In one implementation, the N target regions are pairwise distinct. In one implementation, at least two of the N target genes are distinct from each other. In one implementation, at least two of the N target species are distinct from each other.

[0013] In one implementation, two or more target regions target the same target gene. In another implementation, two or more target regions target the same target species.

[0014] On one hand, the present invention provides a method for detecting pathogens, which uses the positive reference template and / or the reference primers of the present invention, such as the kit of the present invention. The method of the present invention can prevent false positive results in qPCR detection of pathogens caused by contamination of the test sample by a positive control.

[0015] In a fourth aspect, the present invention provides a method for determining the presence of a target region in a test sample derived from a host species, comprising: simultaneously performing real-time quantitative PCR on a test tube and a positive control tube. The test tube contains a test sample with the 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 contains a positive reference material comprising 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. The positive reference polynucleotide includes a reference region corresponding to the target region. The target region includes at least a target upstream primer binding region and a target downstream primer binding region, as well as a target probe binding region between them. The sequence of the target region is identical to or reverse complementary to the sequence of the target segment of the target gene of the target species. The target upstream primer, whose sequence is identical to or contains the sequence of the target upstream primer binding region at its 3' end, and the target downstream primer, whose sequence is reverse complementary to or contains the reverse complementary sequence of the target downstream primer binding region at its 3' end, together can amplify the target region using the target region or a polynucleotide containing the target region as a template. The target probe, whose sequence is identical to or reverse complementary to the target probe binding region, can hybridize with the target region or its amplification product. The reference region is defined by using a target upstream primer binding region in the target region with a sequence that is different (e.g., at least 4, 6, or 8 consecutive nucleotides different at the 3' end, e.g., 10-50 consecutive nucleotides, e.g., 15-40). The target downstream primer binding region may be replaced with a reference upstream primer binding region that is different from the target downstream primer binding region by a sequence ... The reference upstream primer, whose sequence is the same as the reference upstream primer binding region or whose 3' end contains the sequence of the reference upstream primer binding region, and the reference downstream primer whose sequence is reverse complementary to the reference downstream primer binding region or whose 3' end contains the reverse complementary sequence of the reference downstream primer binding region, together can amplify the reference region using the reference region or a polynucleotide containing the reference region as a template.

[0016] In a fifth aspect, the present invention provides a method for determining the presence of N target regions in a test sample derived from a host species, where N is a natural number greater than 2, comprising: simultaneously performing real-time quantitative PCR on N test tubes and N positive control tubes. The nth test tube contains a test sample with the test nucleic acid as a template, the nth target upstream primer and the nth target downstream primer as primer pairs, and the nth target probe as a probe. The nth positive reference tube contains a positive reference material 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 includes an nth reference region, which corresponds to the nth target region. The nth target region includes at least an nth target upstream primer binding region and an nth target downstream primer binding region, as well as an nth target probe binding region between them. The sequence of the nth target region is identical to or reverse complementary to the sequence of the nth target segment of the nth target gene of the nth target species. The nth target upstream primer and its sequence are reverse complementary to the nth target downstream primer binding region, provided their sequences are identical to or contain the sequence of the nth target upstream primer binding region at their 3' ends. The nth target downstream primer, whose 3' end contains the inverse complementary sequence of the nth target downstream primer binding region, together with the nth target region, can amplify the nth target region using the nth target region or a polynucleotide containing the nth target region as a template. The nth target probe, whose sequence is the same as or inversely complementary to the nth target probe binding region, can hybridize with the nth target region or its amplification product. The nth reference region is obtained by differentiating the nth target upstream primer binding region in the nth target region with a sequence different (e.g., at least 4, 6, or 8 consecutive nucleotides at the 3' end, for example, 10-50 consecutive nucleotides). For example, 15-40 consecutive nucleotides, 20-30 consecutive nucleotides, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides different, replacing and / or replacing the nth target downstream primer binding region in the nth target region with a sequence different (e.g., at least 4, 6, or 8 consecutive nucleotides different at the 5' end, for example, 10-50 consecutive nucleotides, 15-40 consecutive nucleotides, 20-30 consecutive nucleotides, or 20, 21, 22, 23, 24, 25...). The nth reference upstream primer is obtained by replacing the binding region of the nth reference upstream primer with the same sequence as the binding region of the nth reference upstream primer or the sequence of the nth reference upstream primer containing the sequence of the nth reference upstream primer at its 3' end. The nth reference downstream primer and the nth reference downstream primer together can amplify the nth reference region using the nth reference region or a polynucleotide containing the nth reference region as a template. n is a natural number from 1 to N.

[0017] In one embodiment, the N positive reference polynucleotides comprise N reference regions, and the nth positive reference polynucleotide comprises the nth reference region. In another embodiment, the N reference regions are contained within a single positive reference polynucleotide.

[0018] In one implementation, the N target regions are pairwise distinct. In one implementation, at least two of the N target genes are distinct from each other. In one implementation, at least two of the N target species are distinct from each other.

[0019] In one implementation, two or more target regions target the same target gene. In another implementation, two or more target regions target the same target species.

[0020] On the one hand, the present invention provides a kit comprising the positive reference template of the present invention and / or the reference primer of the present invention, and optionally, other reagents required for performing qPCR for detecting pathogens.

[0021] In a sixth aspect, the present invention provides a kit comprising: (1) The polynucleotide of the first aspect of the present invention; (2) A polynucleotide according to the second aspect of the present invention; or (3) A set of polynucleotides of the third aspect of the present invention.

[0022] In a seventh aspect, the present invention provides a kit comprising: reagents for carrying out the methods of the fifth or sixth aspect of the present invention.

[0023] On the one hand, the present invention provides oligonucleotides, which are used as reference primers for amplifying the positive reference template of the present invention.

[0024] In one embodiment, the oligonucleotide is at least 8 consecutive nucleotides in length, such as 10-50 consecutive nucleotides, such as 15-40 consecutive nucleotides, such as 20-30 consecutive nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides.

[0025] In one embodiment, the oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides that are not present in the target segment.

[0026] In one embodiment, the oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides that are not present in the target gene.

[0027] In one embodiment, the oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides that are not present in the target species.

[0028] In one embodiment, the oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides that are not present in the host species.

[0029] In one embodiment, the oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides that are not found in nature.

[0030] In one embodiment, the sample is a biological sample derived from the host species. In one embodiment, the sample contains nucleic acids. In one embodiment, the sample contains nucleic acids derived from the target species. In one embodiment, the sample contains nucleic acids originating from the target species. In one embodiment, the sample contains nucleic acids derived from the host species. In one embodiment, the sample contains nucleic acids originating from the host species.

[0031] In one embodiment, the host species is an economically valuable species, i.e., a biological species with economic value. In one embodiment, the host species is a farmed species, such as livestock, poultry, or aquatic animals. In one embodiment, the host species is a cow, goat, sheep, pig, fish, or shrimp. In one embodiment, the host species is a human.

[0032] In one embodiment, the target species is a pathogen that invades a host species. In one embodiment, the target species is African swine fever virus. In one embodiment, the target species is porcine reproductive and respiratory syndrome virus (PRRSV). In one embodiment, the target species is porcine epidemic diarrhea virus (PEDV). In one embodiment, the target species is a porcine coronavirus, such as porcine Delta coronavirus. In one embodiment, the target species is a porcine rotavirus, such as porcine rotavirus group A. In one embodiment, the target species is one or more (e.g., any two or all three) of porcine epidemic diarrhea virus, porcine coronavirus (e.g., porcine Delta coronavirus), and porcine rotavirus (e.g., porcine rotavirus group A). ​​In one embodiment, the target species is shrimp enterocytozoon. In one embodiment, the target species is a Vibrio species (e.g., Vibrio parahaemolyticus, Vibrio Erwinii, Vibrio harveyi, and Vibrio campbellii) carrying the PirA virulence gene (e.g., pVA1 virulence plasmid). In one embodiment, the target species is shrimp infectious hypodermal and hematopoietic necrosis virus. In one embodiment, the target species is *Vibrio parahaemolyticus* (without carrying the PirA and PirB virulence genes). In another embodiment, the target species is one or more of the following (e.g., any two, any three, or all four): *Enterocera hepatocellulare*, *Vibrio parahaemolyticus* carrying the PirA virulence gene (e.g., the pVA1 virulence plasmid), *Vibrio parahaemolyticus*, *Vibrio erwinii*, *Vibrio harveyi*, and *Vibrio campei*, *Vibrio parahaemolyticus*, and *Vibrio parahaemolyticus* (without carrying the PirA and PirB virulence genes). In one embodiment, the target species is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0033] In one embodiment, the target gene is a target species-related gene. In one embodiment, the target gene is a target species-specific gene. In one embodiment, the target gene is a disease-related gene. In one embodiment, the target gene is a pathogenic gene. In one embodiment, the target gene is the B646L gene of African swine fever virus. In one embodiment, the target gene is the ORF7 (N gene) and / or ORF6 (M gene) of porcine reproductive and respiratory syndrome virus. In one embodiment, the target gene is the M gene of porcine epidemic diarrhea virus. In one embodiment, the target gene is the N gene of porcine coronavirus (e.g., porcine Delta coronavirus). In one embodiment, the target gene is the VP6 gene of porcine rotavirus (e.g., porcine rotavirus group A). ​​In one embodiment, the target gene is one or more (e.g., any two or all three) of the M gene of porcine epidemic diarrhea virus, the N gene of porcine coronavirus (e.g., porcine Delta coronavirus), and the VP6 gene of porcine rotavirus (e.g., porcine rotavirus group A). ​​In one embodiment, the target gene is the 18S rRNA of Enterocytozoon hepatocellular carcinoma. In one embodiment, the target gene is the PirA virulence gene of a Vibrio species (e.g., Vibrio parahaemolyticus, Vibrio erwinii, Vibrio harveyi, and Vibrio campeii) carrying the PirA virulence gene (e.g., the pVA1 virulence plasmid). In one embodiment, the target gene is the ORF1 gene of shrimp infectious hypodermal and hematopoietic necrosis virus. In one embodiment, the target gene is the Toxr gene of Vibrio parahaemolyticus (without carrying the PirA and PirB virulence genes). In one embodiment, the target gene is one or more of the following: 18S rRNA of *Enterocera hepatocellular carcinoma*, the PirA virulence gene of Vibrio (e.g., *Vibrio parahaemolyticus*, *Vibrio erwinii*, *Vibrio harveyi*, and *Vibrio campbellii* carrying the PirA virulence gene (e.g., the pVA1 virulence plasmid), the ORF1 gene of shrimp infectious hypodermal and hematopoietic necrosis virus, and the Toxr gene of *Vibrio parahaemolyticus* (without carrying the PirA and PirB virulence genes) (e.g., any two, any three, or all four). In one embodiment, the target gene is one or more of the following: ORF1 (i.e., the ab gene), N gene, and RNase P gene of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2).

[0034] In one embodiment, the disease is African swine fever. In one embodiment, the disease is porcine reproductive and respiratory syndrome (PRRS). In one embodiment, the disease is porcine epidemic diarrhea (PED). In one embodiment, the disease is porcine coronavirus disease, such as porcine Delta coronavirus disease. In one embodiment, the disease is porcine rotavirus disease, such as porcine group A rotavirus disease. In one embodiment, the disease is one or more (e.g., any two or all three) of porcine epidemic diarrhea, porcine coronavirus disease (e.g., porcine Delta coronavirus disease), and porcine rotavirus disease (e.g., porcine group A rotavirus disease). In one embodiment, the disease is shrimp enterocytozoonosis. In one embodiment, the disease is shrimp acute hepatopancreatic necrosis disease. In one embodiment, the disease is shrimp infectious hypodermal and hematopoietic necrosis disease. In one embodiment, the disease is shrimp parahaemolyticus infection. In one embodiment, the disease is one or more (e.g., any two, any three, or all four) of shrimp enterocytozoonosis, shrimp acute hepatopancreatic necrosis disease, shrimp infectious hypodermal and hematopoietic necrosis disease, and shrimp parahaemolyticus infection. In one implementation, the disease is COVID-19 or Severe Acute Respiratory Syndrome.

[0035] In one implementation, the target segment is a characteristic segment in the target gene.

[0036] In one embodiment, the target region is at least 25 consecutive nucleotides in length, such as 30-300 consecutive nucleotides, such as 40-200 consecutive nucleotides, such as 50-100 consecutive nucleotides, such as about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 consecutive nucleotides. "About" means within ±5, ±4, ±3, ±2, or ±1.

[0037] In one embodiment, the length of the reference region is at least 25 consecutive nucleotides, such as 30-300 consecutive nucleotides, such as 40-200 consecutive nucleotides, such as 50-100 consecutive nucleotides, such as about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 consecutive nucleotides. "About" means within ±5, ±4, ±3, ±2, or ±1.

[0038] In one embodiment, the target upstream primer (binding region) is at least 8 consecutive nucleotides in length, for example, 10-50 consecutive nucleotides, for example, 12-35 consecutive nucleotides, for example, 15-25 consecutive nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides. In one embodiment, the sequence of the target upstream primer (binding region) is specific, for example, specific to the target gene or specific to the target species.

[0039] In one embodiment, the target downstream primer (binding region) is at least 8 consecutive nucleotides in length, for example, 10-50 consecutive nucleotides, 12-35 consecutive nucleotides, 15-25 consecutive nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides. In one embodiment, the sequence of the target downstream primer (binding region) is specific, for example, specific to the target gene or specific to the target species.

[0040] In one embodiment, the target probe (binding region) is at least 8 consecutive nucleotides in length, for example, 10-50 consecutive nucleotides, 15-40 consecutive nucleotides, 20-30 consecutive nucleotides, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides. In one embodiment, the sequence of the target probe (binding region) is specific, for example, specific to a target gene or specific to a target species.

[0041] In one embodiment, the target probe is labeled. In one embodiment, the target probe is fluorescently labeled. In one embodiment, the target probe is labeled with a fluorescent group and a quencher group. In one embodiment, the fluorescent group is FAM, VIC, HEX, CY5, or ROX. In one embodiment, the quencher group is BHQ1, BHQ2, BHQ3, TAMRA, or MGB. In one embodiment, the target probe is labeled with the fluorescent group FAN and the quencher group BHQ1. In one embodiment, the target probe is labeled with the fluorescent group VIC and the quencher group BHQ1. In one embodiment, the target probe is labeled with the fluorescent group FAM and the quencher group TAMRA. In one embodiment, the target probe is labeled with the fluorescent group FAM and the quencher group MGB. In one embodiment, the target probe is labeled with the fluorescent group ROX and the quencher group BHQ2. In one embodiment, the target probe is labeled with the fluorescent group CY5 and the quencher group BHQ3. In one embodiment, the target probe is labeled with the fluorescent group VIA and the quencher group BHQ2.

[0042] In one embodiment, the reference primer (binding region) is at least 8 consecutive nucleotides in length, such as 10-50 consecutive nucleotides, such as 15-40 consecutive nucleotides, such as 20-30 consecutive nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides.

[0043] In one embodiment, the reference upstream primer (binding region) is at least 8 consecutive nucleotides in length, such as 10-50 consecutive nucleotides, such as 15-40 consecutive nucleotides, such as 20-30 consecutive nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides.

[0044] In one embodiment, the reference downstream primer (binding region) is at least 8 consecutive nucleotides in length, such as 10-50 consecutive nucleotides, such as 15-40 consecutive nucleotides, such as 20-30 consecutive nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides.

[0045] In one embodiment, the 5' end of the target primer (e.g., the target upstream primer and / or the target downstream primer) contains one or more consecutive nucleotides that do not match the corresponding position of the target region or target segment (i.e., are not identical to one strand and are not complementary to another strand), such as 1-100 consecutive nucleotides, such as 10-50 consecutive nucleotides, such as 15-40 consecutive nucleotides, such as 20-30 consecutive nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides.

[0046] In one embodiment, the 5' end of the reference primer (e.g., a reference upstream primer and / or a reference downstream primer) contains one or more consecutive nucleotides that do not match the corresponding position of the reference region (optionally, and the target region or target segment) (i.e., are not identical to one strand and not complementary to another strand), such as 1-100 consecutive nucleotides, such as 10-50 consecutive nucleotides, such as 15-40 consecutive nucleotides, such as 20-30 consecutive nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides.

[0047] In one embodiment, the target upstream primer binding region is directly linked to the target probe binding region. In another embodiment, the target upstream primer binding region and the target probe binding region are linked by one or more consecutive nucleotides, such as 1-200 consecutive nucleotides, 5-100 consecutive nucleotides, 10-50 consecutive nucleotides, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 consecutive nucleotides. "About" means within ±5, ±4, ±3, ±2, or ±1.

[0048] In one embodiment, the target downstream primer binding region is directly linked to the target probe binding region. In another embodiment, the target downstream primer binding region and the target probe binding region are linked by one or more consecutive nucleotides, such as 1-200 consecutive nucleotides, 5-100 consecutive nucleotides, 10-50 consecutive nucleotides, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 consecutive nucleotides. "About" means within ±5, ±4, ±3, ±2, or ±1.

[0049] In one embodiment, the reference upstream primer binding region is directly linked to the target probe binding region. In another embodiment, the reference upstream primer binding region and the target probe binding region are linked by one or more consecutive nucleotides, such as 1-200 consecutive nucleotides, 5-100 consecutive nucleotides, 10-50 consecutive nucleotides, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 consecutive nucleotides. "About" means within ±5, ±4, ±3, ±2, or ±1.

[0050] In one embodiment, the reference downstream primer binding region is directly linked to the target probe binding region. In another embodiment, the reference downstream primer binding region and the target probe binding region are linked by one or more consecutive nucleotides, such as 1-200 consecutive nucleotides, 5-100 consecutive nucleotides, 10-50 consecutive nucleotides, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 consecutive nucleotides. "About" means within ±5, ±4, ±3, ±2, or ±1.

[0051] In one implementation, N is at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3 or 2.

[0052] In one embodiment, the polynucleotide sequence of the present invention (used as a positive reference template) comprises at least eight consecutive nucleotides, such as 10-50, 12-35, 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, from the 3' end of the oligonucleotide sequence of the present invention (used as a reference primer). In another embodiment, the polynucleotide sequence of the present invention (used as a positive reference template) comprises at least eight consecutive nucleotides, such as 10-50, 12-35, 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, from the 5' end of the reverse complementary sequence of the oligonucleotide sequence of the present invention (used as a reference primer).

[0053] On the other hand, the present invention provides oligonucleotides that are used as reference primers for amplifying the positive reference template of the present invention.

[0054] In another aspect, the present invention provides an oligonucleotide comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or all 25 consecutive nucleotides of the sequence shown in SEQ ID NO: 7 or its reverse sequence, complementary sequence, or reverse complementary sequence. In another aspect, the present invention provides an oligonucleotide substantially composed of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or all 25 consecutive nucleotides of the sequence shown in SEQ ID NO: 7 or its reverse sequence, complementary sequence, or reverse complementary sequence. In one aspect, the present invention provides an oligonucleotide comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or all 25 consecutive nucleotides of the sequence shown in SEQ ID NO: 7 or its reverse sequence, complementary sequence or reverse complementary sequence.

[0055] In another aspect, the present invention provides an oligonucleotide comprising the sequence shown in SEQ ID NO: 7 or its reverse sequence, complementary sequence, or reverse complementary sequence. In another aspect, the present invention provides an oligonucleotide substantially composed of the sequence shown in SEQ ID NO: 7 or its reverse sequence, complementary sequence, or reverse complementary sequence. In yet another aspect, the present invention provides an oligonucleotide composed of the sequence shown in SEQ ID NO: 7 or its reverse sequence, complementary sequence, or reverse complementary sequence.

[0056] In one embodiment, the oligonucleotide is used as a reference primer for amplifying the positive reference template of the present invention. In one embodiment, the oligonucleotide is used as a reference upstream primer. In one embodiment, the oligonucleotide is used as a reference downstream primer. In one embodiment, the oligonucleotide is used as a reference primer for single detection. In one embodiment, the oligonucleotide is used as a reference primer for multiplex detection (i.e., multiple detection items share one primer).

[0057] In one embodiment, the oligonucleotide is as described above with respect to the reference primer and / or the upstream primer and / or the downstream primer.

[0058] On the other hand, the present invention provides a polynucleotide that can be used as a positive reference template in place of a conventional positive control template.

[0059] In another aspect, the present invention provides a polynucleotide comprising a sequence as shown in any of SEQ ID NO: 25, 27, 29, 31, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98, 99, 100, 101. In one embodiment, the polynucleotide is used as a positive reference template of the present invention.

[0060] In another aspect, the present invention provides a polynucleotide comprising one or more sequences as shown in any of SEQ ID NO: 25, 27, 29, 31. In one embodiment, the polynucleotide is used as a positive reference template for detecting African swine fever virus.

[0061] In another aspect, the present invention provides a polynucleotide comprising any one or both of the sequences shown in either SEQ ID NO: 88 or 89. In one embodiment, the polynucleotide is used as a positive reference template for detecting porcine reproductive and respiratory syndrome virus.

[0062] In another aspect, the present invention provides a polynucleotide comprising any one, any two, or all three of the sequences shown in any of SEQ ID NO: 90, 91, 92. In one embodiment, the polynucleotide is used as a positive reference template for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0063] In another aspect, the present invention provides a polynucleotide comprising any one, any two, or all three of the sequences shown in any of SEQ ID NO: 94, 95, and 96. In one embodiment, the polynucleotide is used as a positive reference template for detecting pathogens associated with swine diarrheal diseases, such as porcine epidemic diarrhea virus, porcine Delta coronavirus (e.g., swine coronavirus), and porcine rotavirus (e.g., group A rotavirus). In one embodiment, the polynucleotide further comprises the sequence shown in SEQ ID NO: 97.

[0064] In another aspect, the present invention provides a polynucleotide comprising any one, any two, any three, or all four sequences as shown in any of SEQ ID NO: 98, 99, 100, 101. In one embodiment, the polynucleotide is used as a positive reference template for detecting shrimp disease-related pathogens, such as *Enterocera hepatocellular carcinoma*, *Vibrio parahaemolyticus* carrying the PirA virulence gene (e.g., pVA1 virulence plasmid) (e.g., *Vibrio parahaemolyticus*, *Vibrio erwinii*, *Vibrio harveyi*, and *Vibrio campei*), shrimp infectious hypodermal and hematopoietic necrosis virus, and one or more (e.g., any two, any three, or all four) of *Vibrio parahaemolyticus* (without carrying the PirA and PirB virulence genes).

[0065] In another aspect, the present invention provides a polynucleotide comprising segment a as shown in SEQ ID NO: 88 and segment b as shown in SEQ ID NO: 89. In one embodiment, segment a as shown in SEQ ID NO: 88 is located at the 5' end, and segment b as shown in SEQ ID NO: 89 is located at the 3' end. In another embodiment, segment a as shown in SEQ ID NO: 88 is located at the 3' end, and segment b as shown in SEQ ID NO: 89 is located at the 5' end. In one embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 40. In one embodiment, the polynucleotide is used as a positive reference template for detecting porcine reproductive and respiratory syndrome virus (PRRSV). In one embodiment, the polynucleotide is used as a positive reference template for detecting PRSV.

[0066] In another aspect, the present invention provides a polynucleotide comprising segment a as shown in SEQ ID NO: 90, segment b as shown in SEQ ID NO: 91, and segment c as shown in SEQ ID NO: 92. In one embodiment, segment a as shown in SEQ ID NO: 90 is located at the 5' end, and segment c as shown in SEQ ID NO: 92 is located at the 3' end. In one embodiment, segment a as shown in SEQ ID NO: 90 is located at the 3' end, and segment c as shown in SEQ ID NO: 92 is located at the 5' end. In one embodiment, segment a as shown in SEQ ID NO: 90 is located at the 5' end, and segment b as shown in SEQ ID NO: 91 is located at the 3' end. In one embodiment, segment a as shown in SEQ ID NO: 90 is located at the 3' end, and segment b as shown in SEQ ID NO: 91 is located at the 5' end. In one embodiment, segment c as shown in SEQ ID NO: 92 is located at the 5' end, and segment b as shown in SEQ ID NO: 91 is located at the 3' end. In one embodiment, segment c of the sequence shown in SEQ ID NO: 92 is located at the 3' end, and segment b of the sequence shown in SEQ ID NO: 91 is located at the 5' end. In one embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 53. In one embodiment, the polynucleotide is used as a positive reference template. In one embodiment, the polynucleotide is used as a positive reference template for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0067] In another aspect, the present invention provides a polynucleotide comprising segment a as shown in SEQ ID NO: 94, segment b as shown in SEQ ID NO: 95, and segment c as shown in SEQ ID NO: 96. In one embodiment, segment a as shown in SEQ ID NO: 94 is located at the 5' end, and segment c as shown in SEQ ID NO: 96 is located at the 3' end. In one embodiment, segment a as shown in SEQ ID NO: 94 is located at the 3' end, and segment c as shown in SEQ ID NO: 96 is located at the 5' end. In one embodiment, segment a as shown in SEQ ID NO: 94 is located at the 5' end, and segment b as shown in SEQ ID NO: 95 is located at the 3' end. In one embodiment, segment a as shown in SEQ ID NO: 94 is located at the 3' end, and segment b as shown in SEQ ID NO: 95 is located at the 5' end. In one embodiment, segment c as shown in SEQ ID NO: 96 is located at the 5' end, and segment b as shown in SEQ ID NO: 95 is located at the 3' end. In one embodiment, segment c, as shown in SEQ ID NO: 96, is located at the 3' end, and segment b, as shown in SEQ ID NO: 95, is located at the 5' end. In one embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 93. In one embodiment, the polynucleotide further comprises segment d, as shown in SEQ ID NO: 97. In one embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 70. In one embodiment, the polynucleotide serves as a positive reference template for detecting pathogens associated with swine diarrheal diseases, such as porcine epidemic diarrhea virus, porcine Delta coronavirus (e.g., swine coronavirus), and porcine rotavirus (e.g., group A rotavirus) (e.g., any two or all three).

[0068] In another aspect, the present invention provides a polynucleotide comprising segment a as shown in SEQ ID NO: 98, segment b as shown in SEQ ID NO: 99, segment c as shown in SEQ ID NO: 100, and segment d as shown in SEQ ID NO: 101. In one embodiment, the polynucleotide comprises segments a, b, c, and d sequentially in a 5' to 3' direction. In one embodiment, the polynucleotide comprises segments a, b, d, and c sequentially in a 5' to 3' direction. In one embodiment, the polynucleotide comprises segments a, c, b, and d sequentially in a 5' to 3' direction. In one embodiment, the polynucleotide comprises segments a, c, d, and b sequentially in a 5' to 3' direction. In one embodiment, the polynucleotide comprises segments a, d, b, and c sequentially in a 5' to 3' direction. In one embodiment, the polynucleotide comprises, in a 5' to 3' orientation, segments a, d, c, and b in sequence. (This text is repeated four times in the original.) In one embodiment, the polynucleotide comprises, in a 5' to 3' orientation, segments c, a, b, and d sequentially. (This text is repeated four times in the original.) In one embodiment, the polynucleotide comprises, in a 5' to 3' direction, segments d, a, c, and b in sequence.In one embodiment, the polynucleotide comprises, in a 5' to 3' orientation, segments d, b, a, and c in sequence. In another embodiment, the polynucleotide comprises, in a 5' to 3' orientation, segments d, b, c, and a in sequence. In yet another embodiment, the polynucleotide comprises, in a 5' to 3' orientation, segments d, c, a, and b in sequence. In yet another embodiment, the polynucleotide comprises, in a 5' to 3' orientation, segments d, c, b, and a in sequence. In yet another embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 87. In one embodiment, the polynucleotide is used as a positive reference template for detecting shrimp disease-associated pathogens, such as *Enterocera hepatocellular carcinoma*, *Vibrio parahaemolyticus* carrying the PirA virulence gene (e.g., pVA1 virulence plasmid) (e.g., *Vibrio parahaemolyticus*, *Vibrio erwinii*, *Vibrio harveyi*, and *Vibrio campbellii*), shrimp infectious hypodermal and hematopoietic necrosis virus, and one or more (e.g., any two, any three, or all four) of *Vibrio parahaemolyticus* (without carrying the PirA and PirB virulence genes).

[0069] In one embodiment, the polynucleotide is used as a positive reference template, replacing a conventional positive control template. In one embodiment, the polynucleotide is a single positive reference template (i.e., containing one reference region). In one embodiment, the polynucleotide is a multiple positive reference template (i.e., containing multiple reference regions). In one embodiment, multiple polynucleotides are used as positive reference templates (e.g., each polynucleotide contains one reference region).

[0070] In one implementation, the polynucleotide is as described above with respect to the positive reference template.

[0071] On the other hand, the present invention provides a kit comprising the positive reference template of the present invention and / or the reference primer of the present invention, and optionally, other reagents required for performing qPCR for detecting pathogens.

[0072] In another aspect, the present invention provides a kit comprising one or more of the following reagents: (1) A positive reference template comprising the sequence shown in SEQ ID NO: 25, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 13, and a probe comprising the sequence shown in SEQ ID NO: 14; and / or (2) A positive reference template comprising the sequence shown in SEQ ID NO: 27, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 16, and a probe comprising the sequence shown in SEQ ID NO: 17; and / or (3) A positive reference template comprising the sequence shown in SEQ ID NO: 29, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 19, and a probe comprising the sequence shown in SEQ ID NO: 20; and / or (4) A positive reference template comprising the sequence shown in SEQ ID NO: 31, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 22, and a probe comprising the sequence shown in SEQ ID NO: 23. In one embodiment, the kit is used to detect African swine fever virus.

[0073] In another aspect, the present invention provides a kit comprising one or both of the following groups of reagents: (1) A positive reference template comprising the sequence shown in SEQ ID NO: 88, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 33, and a probe comprising the sequence shown in SEQ ID NO: 34; and / or (2) A positive reference template comprising the sequence shown in SEQ ID NO: 89, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 37, and a probe comprising the sequence shown in SEQ ID NO: 38. Optionally, a positive reference template comprising the sequence shown in SEQ ID NO: 88 and a positive reference template comprising the sequence shown in SEQ ID NO: 89 are together present as a single positive reference template, and optionally, a sequence shown in SEQ ID NO: 40 is included. In one embodiment, the kit is used to detect porcine reproductive and respiratory syndrome virus.

[0074] In another aspect, the present invention provides a kit comprising one, two, or three of the following three groups of reagents: (1) A positive reference template comprising the sequence shown in SEQ ID NO: 90, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 42, and a probe comprising the sequence shown in SEQ ID NO: 43; and / or (2) A positive reference template comprising the sequence shown in SEQ ID NO: 91, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 46, and a probe comprising the sequence shown in SEQ ID NO: 47; and / or (3) A positive reference template comprising the sequence shown in SEQ ID NO: 92, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 49, and a probe comprising the sequence shown in SEQ ID NO: 50. Optionally, a positive reference template comprising the sequence shown in SEQ ID NO: 90, a positive reference template comprising the sequence shown in SEQ ID NO: 91, and a positive reference template comprising the sequence shown in SEQ ID NO: 92 are together presented as a single positive reference template, and optionally, a sequence shown in SEQ ID NO: 53 is included. In one embodiment, the kit is used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0075] In another aspect, the present invention provides a kit comprising one, two, or three of the following three groups of reagents: (1) A positive reference template comprising the sequence shown in SEQ ID NO: 94, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 55, and a probe comprising the sequence shown in SEQ ID NO: 56; and / or (2) A positive reference template comprising the sequence shown in SEQ ID NO: 95, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 59, and a probe comprising the sequence shown in SEQ ID NO: 60; and / or (3) A positive reference template comprising the sequence shown in SEQ ID NO: 96, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 63, and a probe comprising the sequence shown in SEQ ID NO: 64. Optionally, a positive reference template comprising the sequence shown in SEQ ID NO: 94, a positive reference template comprising the sequence shown in SEQ ID NO: 95, and a positive reference template comprising the sequence shown in SEQ ID NO: 96 are presented together as a single positive reference template, optionally comprising the sequence shown in SEQ ID NO: 93 or the sequence shown in SEQ ID NO: 70. In one embodiment, the kit is used to detect pathogens associated with swine diarrheal diseases, such as porcine epidemic diarrhea virus, porcine Delta coronavirus (e.g., swine coronavirus), and porcine rotavirus (e.g., group A rotavirus) (e.g., any two or all three).

[0076] In another aspect, the present invention provides a kit comprising one, two, three, or four of the following groups of reagents: (1) A positive reference template comprising the sequence shown in SEQ ID NO: 98, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 72, and a probe comprising the sequence shown in SEQ ID NO: 73; and / or (2) A positive reference template comprising the sequence shown in SEQ ID NO: 99, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 76, and a probe comprising the sequence shown in SEQ ID NO: 77; and / or (3) A positive reference template comprising the sequence shown in SEQ ID NO: 100, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 80, and a probe comprising the sequence shown in SEQ ID NO: 81; and / or (4) A positive reference template comprising the sequence shown in SEQ ID NO: 101, and optionally, an upstream primer comprising the sequence shown in SEQ ID NO: 7, a downstream primer comprising the sequence shown in SEQ ID NO: 84, and a probe comprising the sequence shown in SEQ ID NO: 85. Optionally, a positive reference template comprising the sequence shown in SEQ ID NO: 98, a positive reference template comprising the sequence shown in SEQ ID NO: 99, a positive reference template comprising the sequence shown in SEQ ID NO: 100, and a positive reference template comprising the sequence shown in SEQ ID NO: 101 are presented together as a single positive reference template, and optionally, the sequence shown in SEQ ID NO: 87 is also included. In one embodiment, the kit is used to detect shrimp disease-related pathogens, such as shrimp enterocytozoa, vibrio carrying the PirA virulence gene (e.g., pVA1 virulence plasmid) (e.g., Vibrio parahaemolyticus, Vibrio Erwinii, Vibrio harveyi, and Vibrio campei), shrimp infectious hypodermal and hematopoietic necrosis virus, and one or more (e.g., any two, any three, or all four) of shrimp Vibrio parahaemolyticus (without carrying the PirA and PirB virulence genes).

[0077] In one embodiment, the polynucleotide of the present invention (used as a positive reference template) further comprises a reference region for an internal control. In one embodiment, the internal control is a host-associated gene. In one embodiment, the internal control is a host-specific gene. In one embodiment, the internal control is a housekeeping gene. In one embodiment, the internal control is a constitutively expressed gene. In one embodiment, the internal control is a GAPD gene. Attached Figure Description

[0078] Figure 1A The first group of qPCR results in Example 1; Figure 1B Results of the second group of qPCR in Example 1; Figure 1C Results of the third group of qPCR in Example 1; Figure 1D Results of the fourth group of qPCR in Example 1; Figure 2A AC-F1 amplification results in Example 2; Figure 2B AC-F' amplification results in Example 2; Figure 2C AC-F'-2 amplification results in Example 2; Figure 2D AC-F'-3 amplification results in Example 2; Figure 3A Example 5: qPCR results of amplifying the first template using four sets of primers and probes; Figure 3B Example 5: qPCR results of amplifying the second template using four sets of primers and probes; Figure 3CExample 5: qPCR results of amplifying the third template using four sets of primers and probes; Figure 3D Example 5: qPCR results of amplifying the fourth template using four sets of primers and probes; Figure 3E Example 5: qPCR results of amplifying the fifth template using four sets of primers and probes; Figure 3F Example 5: qPCR results of amplification of the sixth template using four sets of primers and probes; Figure 4A qPCR results of the first set of templates in Example 6; Figure 4B qPCR results of the second set of templates in Example 6; Figure 4C qPCR results of the third template in Example 6; Figure 4D qPCR results of the fourth template in Example 6; Figure 4E qPCR results of the fifth template in Example 6; Figure 4F qPCR results of the sixth template group in Example 6; Figure 5A qPCR results of the first set of templates in Example 7; Figure 5B qPCR results of the second set of templates in Example 7; Figure 5C qPCR results of the third template in Example 7; Figure 5D qPCR results of the fourth template in Example 7; Figure 5E qPCR results of the fifth template in Example 7; Figure 5F qPCR results of the sixth template in Example 7; Figure 6A qPCR results of the first set of templates in Example 8; Figure 6B qPCR results of the second set of templates in Example 8; Figure 6C qPCR results of the third template in Example 8; Figure 6D qPCR results of the fourth template in Example 8; Figure 6E qPCR results of the fifth template in Example 8; Figure 6F qPCR results of the sixth template in Example 8; Figure 7A qPCR results of the first set of templates in Example 9; Figure 7B qPCR results of the second set of templates in Example 9; Figure 7C qPCR results of the third template in Example 9; Figure 7D qPCR results of the fourth template in Example 9; Figure 7E qPCR results of the fifth template in Example 9; Figure 7F qPCR results of the sixth template in Example 9. Detailed Implementation

[0079] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application shall be determined by the claims.

[0080] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0081] To avoid false negatives, positive controls are indispensable in commercial qPCR diagnostic kits, monitoring the preparation and operation of the quantitative PCR reaction system. However, false positives caused by aerosol contamination of the positive control are a persistent problem for testing laboratories. This invention addresses this by modifying the positive control sequence to create a positive control that can both perform quality control functions and eliminate aerosol contamination.

[0082] Example 1: Feasibility Verification of Positive Reference Material This embodiment is used to verify that the positive reference material of the present invention can be used as a positive control for quality control qPCR reagent, thus proving the feasibility of the positive reference material of the present invention.

[0083] The positive sample target sequence, i.e., the AC sequence, was designed and synthesized, and ligated to the plasmid backbone to obtain a conventional positive template plasmid containing the positive sample target sequence. Two sets of primers and probes, AC-F1, AC-R1, AC-P1 and AC-F2, AC-R2, AC-P2, were designed targeting two target regions based on the AC sequence. The AC-F1 and AC-F2 sequences in the AC sequence were replaced with the AC-F' sequence to obtain the sequence-modified positive template sequence of this invention, i.e., the ACT sequence. The ACT sequence was ligated to the plasmid backbone to obtain a plasmid containing the sequence-modified positive template. The positive reference mixture of this invention is a mixture of plasmid containing the ACT sequence and the AC-F' primer sequence. AC-F', AC-R1, AC-P1 and AC-F', AC-R2, AC-P2 constitute two sets of amplification primer and probe combinations targeting the ACT template sequence. The DNA sequences are shown in Table 1, where the underlined and bolded sequences are the differences between the AC and ACT sequences, i.e., the parts of the AC sequence that have been modified.

[0084] Using Novizan's qPCR detection reagent Animal Detection U + The Probe qPCR Super PreMix (catalog number QV114) was used, and experiments were conducted according to the protocol recommended in the kit instructions. Four sets of experiments were designed: Set 1 used primers, probes, and templates containing AC-F1, AC-R1, AC-P1, AC-F2, AC-R2, AC-P2, and a plasmid containing the AC sequence; Set 2 used primers, probes, and templates containing AC-F1, AC-R1, AC-P1, AC-F2, AC-R2, AC-P2, and a plasmid containing the ACT sequence; Set 3 used primers, probes, and templates containing AC-F', AC-R1, AC-P1, AC-R2, AC-P2, and a plasmid containing the ACT sequence; Set 4 used primers, probes, and templates containing AC-F', AC-R1, AC-P1, AC-R2, AC-P2, and a plasmid containing the AC sequence. The plasmid template concentration was 1 × 10⁻⁶. -4 The concentration of primers was 10 μM, and the dosage was 5 μL. The primer concentration was 10 μM, and the dosage was 0.3 μL. Each experimental group had three replicates, and replicates with large fluctuations due to operation were discarded.

[0085] Experimental results are as follows Figure 1A-1D As shown, the average CT values ​​of the two targets in the first group were 27.65 and 25.98, respectively, while the second group showed no amplification curve, indicating that the positive template plasmid containing the sequence modification would not contaminate the target reaction system. In the third group, the average CT values ​​of the two targets in the positive reference mixture of this invention were 26.35 and 25.08, respectively, while the fourth group showed no amplification, indicating that the positive reference mixture of this invention can be used to control the qPCR amplification process.

[0086] Table 1: DNA sequences in this embodiment

[0087] Example 2: Selection of F' primer sequence in positive reference sample This embodiment is used to verify the design rules of AC-F´ in the positive reference mixture of the present invention.

[0088] As shown in the above examples, the AC-F' primers, together with AC-R1 / P1 and AC-R2 / P2, amplify the modified positive template sequence, i.e., the ACT sequence. The design of the AC-F' primers should follow primer design principles; the sequence length is generally between 15-30 bases. Primers that are too short will reduce amplification specificity, while primers that are too long will lead to a decrease in hybridization rate. The Tm value difference between the upstream and downstream primers should ideally not exceed 2°C, and the GC content of the primers should be between 40% and 60%. The AC-F' primers should not pair complementaryly with non-target regions.

[0089] Based on the ACT sequence, this invention designed two additional sets of AC-F' primers, namely AC-F'-2 and AC-F'-3, which together with AC-F' form three sets of F' primers. The base sequences, base lengths, GC content, and Tm values ​​of the three sets of AC-F' primers and AC-R1 primer are shown in Table 2. The plasmid containing the sequence-modified positive template ACT sequence was amplified according to the steps in Example 1, and the amplification function and linear pattern of AC-F1 and the three sets of F' primers were tested.

[0090] Experimental results are as follows Figure 2A-2D The results showed that the CT value for AC-F1 amplifying the AC sequence was 27.65, the CT value for AC-F' amplifying the ACT sequence was 27.28, the CT value for AC-F'-2 amplifying the ACT sequence was 26.86, and the CT value for AC-F'-3 amplifying the ACT sequence was 27.55. This example demonstrates that all three F' primers can successfully amplify the ACT sequence, indicating that F' primers, following primer design principles, can be used as primers in the positive reference mixture of this invention.

[0091] Compared with AC-F1, among the three sets of F' primers, AC-F' and AC-F'-3 amplified the AC sequence with AC-F1 with a ΔCT value within 0.5, while AC-F' and AC-F'-2 amplified the AC sequence with AC-F1 with a typical S-shaped linear pattern. Based on the above, the amplification of AC-F' and AC-F1 is the closest, and subsequent experiments were conducted using the AC-F' sequence.

[0092] Table 2: Information on AC-F1 and the three sets of F' primers

[0093] Example 3: Determination of component dosage in positive reference mixture The positive reference mixture of this invention comprises two components: a sequence-modified positive template plasmid (e.g., the plasmid containing the ACT sequence in Example 1) and an F' primer designed based on the sequence-modified positive template plasmid. The concentration of the plasmid containing the sequence-modified positive template sequence determines the CT value of the positive control; generally, the CT value of the positive control is around 25. The concentration range of the sequence-modified positive template plasmid used in this invention's testing is 1.0 × 10⁻⁶. -4 -1.0×10 -6 ng / μl, CT value ranges from 25 to 30.

[0094] When performing multi-target amplification, multiple positive controls are required. The modified positive template sequences of the present invention for multiple positive controls can be designed on the same plasmid, or multiple positive template plasmids modified with the present invention's sequences can be designed based on the target multiplicity. Similarly, the F' primer sequences designed for different targets and modified positive template sequences can be the same or different. When multiple F' primer sequences are different, the design must not only follow conventional primer design principles but also ensure that different F' primers avoid non-specific amplification. Since a positive control is required for each target sequence amplification, when the F' primer sequences in the positive reference samples of the present invention designed for multiplex amplification are the same, the amount of F' primers in the system will be related to the amplification multiplicity; the higher the amplification multiplicity, the more F' primers are consumed.

[0095] Example 1 illustrates this: the modified positive template sequences for two targets are designed on the same plasmid, and the F' primer sequences are also identical. Therefore, using the two amplification system sequences in Example 1 as examples, the amount of F' primers added in single and double qPCR amplification systems is investigated. The experiments were conducted according to the steps of Example 1, and the input amounts of each component in the single and multiplex systems are shown in Tables 3 and 4.

[0096] Table 3: Amount of Amplification Components Added to Single-System

[0097] Table 4: Amount of Dual-System Amplification Components Added

[0098] Table 5: Test Results of AC-F' Addition Amount

[0099] The experimental CT values ​​are shown in Table 5. The ΔCT value represents the difference between the CT value obtained with different amounts of AC-F' primers and the CT value obtained with 0.3 μl of AC-F' primers. Generally, a ΔCT value within ±0.5 indicates no significant difference in CT values ​​between the two. Table 5 shows that for single amplification, there was no significant difference in CT values ​​between 0.15 μl and 0.3 μl of AC-F' primers. Similarly, for double amplification, there was no significant difference in CT values ​​between 0.2 μl and 0.3 μl of AC-F' primers. Therefore, the amount of AC-F' added is related to the reagent weight; the higher the reagent weight, the more AC-F' should be added.

[0100] The maximum number of amplification reactions used in the embodiments of this invention is four. If the same F' primer is used for all reactions, the maximum amount of F' primer required is 0.4 μl. If the detection reagent is a five-fold or more detection reagent, multiple F' primers can be selected to achieve the same results as in this invention.

[0101] Example 4: Positive reference material simulation aerosol contamination dosage test The positive reference material of this invention can eliminate false positive results caused by aerosol contamination of the positive control. To quantify this phenomenon, we attempted to add a trace amount of the reference material of this invention to the negative sample to simulate aerosol contamination. The amount of positive reference material of this invention added to simulate aerosol contamination was tested.

[0102] Taking the AC-F1, AC-R1, and AC-P1 primer-probe systems as examples, different amounts of the positive reference mixture of this invention were added to the amplification system to simulate aerosol contamination. Three groups of positive reference mixtures of this invention were set up, with the first group consisting of 5 μl of a mixture with a concentration of 1×10⁻⁶. -4 The first mixture consisted of 5 μl of a plasmid containing the ACT sequence (ng / μl) and 0.2 μl of 10 μM AC-F' primers. The second mixture consisted of 5 μl of a 1×10⁻⁶ m² plasmid. -4 The first mixture consisted of ng / μl of plasmid containing the ACT sequence and 0.3 μl of 10 μM AC-F' primers. The second mixture consisted of 5 μl of a 1×10⁻⁶ m³ / μl solution of the first ACT sequence. -4 0.4 μl of 10 μM AC-F' primers and 0.1 μl, 0.2 μl, 0.3 μl, 0.5 μl, and 5 μl of the above three mixtures were added to the amplification system as contaminants in this example.

[0103] The results of the aerosol contamination simulation experiment are shown in Table 6. When the amount of the three positive reference mixtures of this invention as contaminants was 5 μl, the amplification reagent could amplify normally, and there was no significant difference in the sensitivity (CT value) of the amplification of the three mixtures. This indicates that the positive reference mixture at this dose can achieve the quality control function of whether the reagent components can be amplified. When the amount of the three positive reference mixtures of this invention was 0.1 μl, no amplification reaction occurred, indicating that this dose of the positive reference mixture of this invention can eliminate false positive results caused by aerosol contamination of the positive control.

[0104] Patent CN115873663B mentions that "PCR products have a high copy number, generally 10". 13 With a concentration of copies / mL, under continuous thermal cycling conditions, any slight oversight can easily lead to the formation of PCR aerosols... It is estimated that a single aerosol particle may contain 48,000 copies of PCR product, meaning there is approximately an eight-order-of-magnitude gap between aerosol particles and their products. Based on this order-of-magnitude relationship between PCR products and aerosol particles, even with an addition of only 0.1 μl of any of the above-mentioned positive reference samples, the concentration far exceeds the potential contamination level caused by aerosols. Therefore, in the following examples, 0.1 μl of the positive reference sample from this invention is added to the negative sample to simulate aerosol contamination without causing amplification.

[0105] Table 6: Quantitative Test Results of Aerosol Pollution

[0106] Example 5: African Swine Fever Singleton qPCR Detection The capsid protein p72, encoded by the B646L gene, has high immunogenicity and is commonly used for nucleic acid detection of African swine fever virus (ASFV). This embodiment uses primer and probe sequences from four recommended qPCR methods to detect ASFV, and tests the application of the positive reference mixture of this invention in singlet qPCR detection. Specific sequences are shown in Table 7.

[0107] Table 7: Recommended primer and probe sequences for single detection of African swine fever virus

[0108] Using Novizan's qPCR detection reagent Animal Detection U +The Probe qPCR Super PreMix (catalog number QV114) was used according to the recommended protocol in the kit instructions. The following templates were added to the four detection systems: The first template was a real extracted African swine fever virus (ASFV) positive nucleic acid sample, with an input volume of 5 μl. The amplified product sequences were ASFV-A, ASFV-B, ASFV-C, and ASFV-D, respectively. The second template was a real extracted ASFV negative nucleic acid sample, with an input volume of 5 μl. The third template was the positive reference mixture of this invention, i.e., a mixture of an artificial plasmid containing the sequence-modified positive template sequence and upstream AC-F' primers designed for that sequence (i.e., the AC-F' primers in Example 1). The artificial plasmid in the first primer / probe (AF, AR, and AP) system contained the ASFV-AT sequence, the artificial plasmid in the second primer / probe system contained the ASFV-BT sequence, and so on. The template input volume was 4.8 μl, representing a concentration of 1 × 10⁻⁶. -5 The experiment consisted of three sets of templates: ng / μl of artificial plasmid and 0.2 μl of 10 μM AC-F' primers; ddH2O was used as the template in the fourth set; a mixture of 5 μl of real extracted African swine fever virus positive nucleic acid sample and the positive reference material of the present invention was used as the template in the third set of experiments (i.e., 0.1 μl of the positive reference material mixture from the third set of experiments was used as an aerosol pollutant); and a mixture of 5 μl of real extracted African swine fever virus negative nucleic acid sample and the positive reference material of the present invention was used as the template in the sixth set of experiments (i.e., 0.1 μl of the positive reference material mixture from the third set of experiments was used as an aerosol pollutant). Each experiment was performed in triplicate, and the average value of the results was taken. Table 8 shows the target sequences amplified by the four sets of primers and probes and the positive template sequences of the present invention obtained by modifying these target sequences. The underlined and bolded sequences are the distinguishing sequences between the two, that is, the modifications made to the positive template of the present invention.

[0109] Table 8: Target sequences amplified by four sets of primers and probes and modified positive template sequences

[0110] The experimental results are shown in Figures 3A-3FThe CT values ​​for the first group of experiments were 30.97, 30.55, 30.92, and 31.63, respectively; the second group of negative samples showed negative amplification results; the third group of experiments contained AC-F' primers complementary to the sequence-modified positive template, and the CT values ​​for qPCR were 27.46, 27.07, 27.58, and 28.35, indicating that the positive reference sample could be amplified normally, and the amplification curves were normal (e.g., uneven curves, disordered curves, no amplification curve, end-jumping curves, etc.); the fourth group of experiments used ultrapure water as the template, and the amplification... The results were negative. The fifth group of experiments added 0.1 μl of the positive reference mixture of the present invention to the first group of experiments. The CT values ​​were 30.84, 30.50, 31.14, and 31.79, respectively. There was no significant difference compared with the CT values ​​of the first group of experiments, indicating that the positive reference of the present invention does not affect the experimental results of real positive samples. The sixth group of experiments added 0.1 μl of the positive reference mixture of the present invention to the second group of experiments. The amplification results were still negative, indicating that the positive reference of the present invention does not affect the experimental results of real negative samples.

[0111] Example 6: Dual RT-qPCR detection of porcine reproductive and respiratory syndrome virus Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that can be detected by RT-qPCR. This example uses this virus as an example to design primer probes for PRRSV from two sources in Europe and America. At the same time, a positive reference mixture of the present invention is designed. The artificial plasmid in the mixture contains the modified positive template sequence of PRRSV from two sources in Europe and America, namely the PRRSV-T sequence, and the upstream primers of the two target regions on the PRRSV-T sequence are both AC-F' primers (that is, the AC-F' primers in Example 1). This embodiment is used to verify whether the positive reference material of the present invention will affect the experimental results of the real sample in the double detection. The required primers, probes and sequence-modified positive template sequences are shown in Table 9. PRRSV-EU is the amplification product of PRRSV-EU-F, PRRSV-EU-R and PRRSV-EU-P (for ORF7, i.e. N gene), and PRRSV-NA is the amplification product of PRRSV-NA-F, PRRSV-NA-R and PRRSV-NA-P (for ORF6, i.e. M gene). The underlined and bolded sequences are the distinguishing sequences between PRRSV-EU, PRRSV-NA and PRRSV-T sequences.

[0112] Table 9: Primer, probe, and amplification product sequences in PRRSV dual detection

[0113] Note: The base R represents adenine (A) and guanine (G), and the base T represents cytosine (C) and thymine (T). Using Novizan's RT-qPCR detection reagent AccurSTART II U + The One Step RT-qPCR Probe Kit (for Fast) (catalog number Q233) was used according to the recommended protocol in the kit instructions. The following templates were added to the test reagents: The first template was a real extracted porcine reproductive and respiratory syndrome virus (PRRSV) double-positive nucleic acid sample (European and American versions), with an input volume of 5 μl. The amplified product sequences were PRRSV-EU and PRRSV-NA sequences. The second template was a real extracted porcine reproductive and respiratory syndrome virus (PRRSV) double-negative nucleic acid sample (European and American versions), with an input volume of 5 μl. The third template was the positive reference mixture of this invention, i.e., a mixture of an artificial plasmid containing the sequence-modified PRRSV-T sequence and upstream AC-F' primers designed for its sequence, wherein the template input volume was 4.8 μl, representing a concentration of 1 × 10⁻⁴. -5 The experiment consisted of ng / μl of artificial plasmid and 0.2 μl of 10 μM PRRSV-F' primers; the template for the fourth group of experiments was ddH2O; the template for the fifth group was a mixture of real extracted porcine reproductive and respiratory syndrome virus (PRRSV) double-positive nucleic acid samples from Europe and the United States and the positive reference material of this invention, wherein the amount of real extracted PRSV double-positive nucleic acid samples from Europe and the United States added was 5 μl, and the amount of the positive reference material mixture of this invention added was 1 / 50 of the amount of template added in the third group of experiments, i.e., 0.1 μl of the positive reference material mixture of this invention from the third group of experiments was taken as aerosol pollutant; the template for the sixth group was a mixture of real extracted porcine negative nucleic acid samples and the positive reference material of this invention, wherein the amount of real extracted negative nucleic acid samples added was 5 μl, and the amount of the positive reference material mixture of this invention added was 1 / 50 of the amount of template added in the third group of experiments, i.e., 0.1 μl of the positive reference material mixture of this invention from the third group of experiments was taken as aerosol pollutant. Each experiment was performed in triplicate, and the average value of the results was taken.

[0114] The experimental results are shown in Figures 4A-4FThe results of the experiments were as follows: The first group of experiments showed CT values ​​of 31.51 and 33.23 for European and American viruses, respectively; the second group of experiments showed negative amplification results; the third group of experiments used the positive reference mixture of this invention, with CT values ​​of 27.13 and 28.34 for European and American viruses, indicating that the positive reference mixture could be used for normal PCR amplification and the amplification curve was normal; the fourth group of experiments showed negative amplification results because the template was ultrapure water; the fifth group of experiments added 0.1 μl of the positive reference mixture of this invention to positive samples, with CT values ​​of 31.69 and 33.36, respectively, showing no significant difference from the CT values ​​of the first group of experiments, indicating that the positive reference mixture of this invention does not affect the experimental results of real positive samples; the sixth group of experiments added 0.1 μl of the positive reference mixture of this invention to negative samples, with no amplification, indicating that the positive reference mixture of this invention does not affect the experimental results of real negative samples.

[0115] Example 7: Detection of 2019-Novel Coronavirus Triple RT-qPCR The 2019-nCoV virus was detected using RT-qPCR technology. Three target regions were designed, and a positive reference mixture was also designed. The artificial plasmid in the mixture contained three modified positive template sequences of the target sequences, namely the nCoV-T sequence. The upstream primers for the three target regions on the nCoV-T sequence were all AC-F' primers (i.e., the AC-F' primers in Example 1). This example was used to verify whether the positive reference of the present invention would affect the experimental results of real samples in triple detection. The required primers, probes, and amplification product sequences are shown in Table 10. The underlined and bolded sequences are the distinguishing sequences between the nCoV-ORF1 (ORF1, ab gene), nCoV-N (N gene), nCoV-IPC (RNase P gene), and nCoV-T sequences.

[0116] Table 10: Primer, probe, and product sequences in the triple amplification of the 2019-Novel Coronavirus

[0117] Using Novizan's RT-qPCR detection reagent AccurSTART II U +The One Step RT-qPCR Probe Kit (for Fast) (catalog number Q233) was used according to the recommended protocol in the kit instructions. The following templates were added to the test reagents: The first template was 5 μl of real extracted SARS-CoV-2 positive sample nucleic acid, with amplification products including nCoV-ORF1, nCoV-N, and nCoV-IPC sequences; the second template was 5 μl of real extracted SARS-CoV-2 negative sample nucleic acid; the third template was the positive reference mixture of this invention, i.e., a mixture containing an artificial plasmid with a modified nCoV-T sequence and upstream AC-F' primers designed against its sequence, with a template dosage of 4.7 μl of 1×10⁻⁶. - 5 The test included ng / μl of artificial plasmid and 0.3 μl of 10 μM AC-F' primers; the fourth template was ddH2O; the fifth template was a mixture of real extracted COVID-19 positive sample nucleic acid and the positive reference material of this invention, wherein the amount of real extracted COVID-19 positive sample nucleic acid added was 5 μl, and the amount of the positive reference material mixture of this invention added was 1 / 50 of the amount of template added in the third experiment, i.e., 0.1 μl of the positive reference material mixture of this invention from the third experiment was taken as an aerosol pollutant; the sixth template was a mixture of real extracted COVID-19 negative sample nucleic acid and the positive reference material of this invention, wherein the amount of real extracted COVID-19 negative sample nucleic acid added was 5 μl, and the amount of the positive reference material mixture of this invention added was 1 / 50 of the amount of template added in the third experiment, i.e., 0.1 μl of the positive reference material mixture of this invention from the third experiment was taken as an aerosol pollutant. Each experiment was performed in triplicate, and the results were averaged.

[0118] The experimental results are shown in Figures 5A-5FIn the first group of experiments, the CT value of the nCoV-ORF1 target was 23.69, the CT value of the nCoV-N gene target was 22.94, and the CT value of the nCoV-IPC internal reference target was 23.35. The second group of experiments showed negative amplification results with no amplification curve. The third group of experiments used the positive reference mixture of this invention, and the CT values ​​of the three modified positive templates were 29.92, 29.32, and 28.56, respectively, indicating that the positive reference mixture of this invention can undergo normal PCR amplification, and the amplification curve is normal. In the fourth group of experiments, the template was ultrapure water. The amplification result was negative, with no amplification curve. In the fifth group of experiments, 0.1 μl of the positive reference mixture of this invention was added to the positive sample. The CT values ​​of the three targets were 23.75, 22.93, and 23.43, respectively, which were not significantly different from the CT values ​​of the first group, indicating that the positive reference of this invention does not affect the experimental results of the real positive sample. In the sixth group of experiments, 0.1 μl of the positive reference mixture of this invention was added to the negative sample. The amplification result was negative, with no amplification curve, indicating that the positive reference of this invention does not affect the experimental results of the real negative sample. (Note: Due to the differences in the automatically set threshold lines of different fluorescence channels, such as FAM, VIC, CY5, etc., the baseline subtraction between channels is inconsistent. Therefore, when the qPCR results of multiple fluorescence channels are displayed in one figure, the amplification curve that enters the logarithmic phase earlier may have a larger CT value.) Example 8: Quadruple RT-qPCR Detection of Porcine Diarrheal Diseases Four targets were detected using RT-qPCR: porcine epidemic diarrhea virus (PEDV) (specifically the M gene), porcine Delta coronavirus (PDCoV) (specifically the N gene), porcine rotavirus group A (PoRV) (specifically the VP6 gene), and porcine internal control (GAPDH). A positive reference mixture was designed, containing an artificial plasmid with modified positive template sequences for the four target sequences, namely the PPPG-T sequence. The upstream primers for the four target regions on the PPPG-T sequence were all AC-F' primers (i.e., the AC-F' primers in Example 1). This example was used to verify whether the positive reference mixture of the present invention would affect the experimental results of real samples in the quadruple detection of porcine diarrheal diseases. The required primers, probes, and amplification product sequences are shown in Table 11. The underlined and bolded sequences are the distinguishing sequences between PEDV-C, PDCoV-C, PoRV-C, GAPDH-C, and PPPG-T sequences.

[0119] Table 11: Primers, probes, and product sequences for quadruple detection of swine diarrhea

[0120] The AccurSTART II U+ One Step RT-qPCR Probe Kit (for Fast) (catalog number Q233) from Novizan was used, and experiments were conducted according to the protocol recommended in the kit instructions. The first experimental template consisted of a real-extracted positive mixed nucleic acid sample of porcine epidemic diarrhea virus (PEDV), porcine Delta coronavirus (PDCoV), and porcine rotavirus, containing the GAPDH internal reference gene. The input volume was 5 μl, and the amplification products were PEDV-C, PDCoV-C, PoRV-C, and GAPDH-C sequences. The second experimental template consisted of a real-extracted negative mixed nucleic acid sample of PEDV, PDCoV, and porcine rotavirus, with an input volume of 5 μl. The third experimental template was a positive reference mixture designed for this example, namely, a mixture containing an artificial plasmid with a modified PPPG-T sequence and upstream AC-F' primers designed for that sequence. The input volume of this mixture was 4.6 μl of a 1×10⁻⁶ concentration. -5 The experimental setup consisted of ng / μl of artificial plasmid and 0.4 μl of 10 μM AC-F' primers; the template for the fourth experiment was ddH2O; the fifth experiment was based on the first experiment with the addition of 0.1 μl of the positive reference mixture from the third experiment; and the sixth experiment was based on the second experiment with the addition of 0.1 μl of the positive reference mixture from the third experiment. Each experiment was performed in triplicate, and the average value was taken.

[0121] The experimental results are shown in Figures 6A-6FIn the first group of experiments, the CT value of porcine epidemic diarrhea virus was 28.43, the CT value of porcine Delta coronavirus was 27.63, the CT value of porcine rotavirus group A was 23.89, and the CT value of porcine internal reference GAPDH gene was 24.37; the amplification result of the second group of experiments was negative; the third group of experiments was a mixture of positive reference materials of the present invention, in which the CT value of the positive control of porcine epidemic diarrhea virus was 26.68, the CT value of the positive control of porcine Delta coronavirus was 26.02, the CT value of the positive control of porcine rotavirus group A was 25.98, and the CT value of the positive control of porcine internal reference GAPDH gene was 26.15, indicating that the positive reference materials of the present invention can be amplified normally by PCR and the amplification curve is normal; The fourth group of experiments used ultrapure water, and the amplification result was negative. The fifth group of experiments added 0.1 μl of the positive reference mixture of this invention to the positive samples. The CT values ​​for porcine epidemic diarrhea virus, porcine Delta coronavirus, porcine rotavirus group A, and the porcine internal reference GAPDH gene were 28.58, 27.69, 23.83, and 24.24, respectively, showing no significant difference from the CT values ​​in the first group of experiments. This indicates that the positive reference mixture of this invention does not affect the experimental results of truly positive samples of porcine diarrheal diseases. The sixth group of experiments added 0.1 μl of the positive reference mixture of this invention to the negative samples, and the amplification result was negative, indicating that the positive reference mixture of this invention does not affect the experimental results of truly negative samples of porcine diarrheal diseases. (Note: Due to differences in the automatically set threshold lines of different fluorescence channels, such as FAM, VIC, and CY5, the baseline subtraction between channels is inconsistent. This results in the phenomenon that when the qPCR results of multiple fluorescence channels are displayed in one graph, the amplification curve that enters the logarithmic phase earlier may have a larger CT value.) Example 9: Quadruple qPCR Detection of Shrimp Diseases The study used qPCR to detect four disease targets: Enterocytozoon hepatisimidae (EHP) (specifically, 18S rRNA), Vibrio parahaemolyticus (EHP) (specifically, 18S rRNA), Vibrio parahaemolyticus (EHP), Vibrio erwinii (EHP), Vibrio harveyi (EHP), and Vibrio campeii (EHP), which carry the PirA virulence gene (e.g., pVA1 virulence plasmid) (specifically, the PirA gene), which causes acute hepatopancreatic necrosis disease (AHPND), Infectious Hypodermic and Hematopoietic Necrosis Virus (IHHNV) (specifically, ORF1), which causes infectious hypodermic and hematopoietic necrosis disease, and Vibrio parahaemolyticus (VP) (specifically, the Toxr gene), which causes vibrio parahaemolytic disease. A positive reference mixture was also designed, containing an artificial plasmid with four modified positive template sequences (EAIV-T sequences) for each target region. The upstream primers for the four target regions on the EAIV-T sequence were all AC-F' primers (i.e., the AC-F' primers in Example 1). This embodiment is used to verify whether the positive reference material of the present invention will affect the experimental results of real samples in the quadruple detection of shrimp diseases. The required primers, probes and amplification product sequences are shown in Table 12. The underlined and bolded sequences are the distinguishing sequences between EHP-C, AHPND-C, IHHNV-C, VP-C and EAIV-T sequences.

[0122] Table 12: Primers, probes, and product sequences for quadruple detection of shrimp diseases

[0123] The Animal Detection U+ Probe qPCR Super PreMix (catalog number QV114) qPCR detection reagent from Novizan was used, and experiments were conducted according to the protocol recommended in the kit instructions. The first experimental template consisted of a 5 μl sample of real-extracted positive mixed nucleic acid samples of shrimp enterocytozoonosis, acute hepatopancreatic necrosis, infectious hypodermal and hematopoietic necrosis, and Vibrio parahaemolyticus. The amplification products were EHP-C, AHPND-C, IHHNV-C, and VP-C sequences. The second experimental template consisted of a 5 μl sample of real-extracted negative mixed nucleic acid samples of shrimp enterocytozoonosis, acute hepatopancreatic necrosis, infectious hypodermal and hematopoietic necrosis, and Vibrio parahaemolyticus. The third experimental template was a positive reference mixture designed for this example, consisting of an artificial plasmid containing the sequence-modified EAIV-T sequence and an upstream AC-F' primer designed for that sequence. The mixture was 4.6 μl at a concentration of 1×10⁻⁶. -6The experimental setup consisted of ng / μl of artificial plasmid and 0.4 μl of 10 μM AC-F' primers; the template for the fourth experiment was ddH2O; the fifth experiment was based on the first experiment with the addition of 0.1 μl of the positive reference mixture from the third experiment; and the sixth experiment was based on the second experiment with the addition of 0.1 μl of the positive reference mixture from the third experiment. Each experiment was performed in triplicate, and the average value was taken.

[0124] The experimental results are shown in Figures 7A-7F In the first group of experiments, the CT values ​​for shrimp enterocytozoonosis were 22.82, for shrimp acute hepatopancreatic necrosis disease 23.19, for shrimp infectious hypodermal and hematopoietic necrosis disease 29.55, and for shrimp Vibrio parahaemolyticus 29.75. The second group of experiments showed negative amplification results. The third group of experiments used a mixture of positive reference materials from this invention, where the CT values ​​for shrimp enterocytozoonosis disease were 26.03, for shrimp acute hepatopancreatic necrosis disease 26.59, for shrimp infectious hypodermal and hematopoietic necrosis disease 25.71, and for shrimp Vibrio parahaemolyticus 26.16, indicating that the positive reference materials of this invention could be normally amplified by PCR, and the amplification curve was normal. The fourth group of experiments used ultrafiltration... The amplification result was negative in pure water. In the fifth group of experiments, 0.1 μl of the positive reference mixture of this invention was added to the nucleic acid of the positive sample. The CT values ​​for *Enterocera hepatis*, *Acute hepatopancreatic necrosis*, *Infectious hypodermal and hematopoietic necrosis*, and *Vibrio parahaemolyticus* were 22.89, 23.10, 29.25, and 29.78, respectively, showing no significant difference from the CT values ​​in the first group of experiments. This indicates that adding the positive reference mixture of this invention does not affect the results of the quadruple qPCR experiment. In the sixth group of experiments, 0.1 μl of the positive reference mixture of this invention was added to the nucleic acid of the negative sample, and the amplification result was negative, indicating that the positive reference mixture of this invention does not affect the experimental results of the true negative sample. (Note: Due to differences in the automatically set threshold lines of different fluorescence channels, such as FAM, VIC, and CY5, the baseline subtraction between channels is inconsistent. This results in the phenomenon that when the qPCR results of multiple fluorescence channels are displayed in one graph, the amplification curve that enters the logarithmic phase earlier may have a larger CT value.)

Claims

1. A polynucleotide comprising a reference region corresponding to a target region, the target region comprising at least a target upstream primer binding region and a target downstream primer binding region and a target probe binding region between them, wherein the sequence of the target region is identical to or reverse complementary to the sequence of a target segment of a target gene of a target species, and the target upstream primer whose sequence is identical to or contains the sequence of the target upstream primer binding region at its 3' end, and the target downstream primer whose sequence is reverse complementary to or contains the reverse complementary sequence of the target downstream primer binding region at its 3' end. The primers together can amplify the target region using the target region or a polynucleotide containing the target region as a template. The target probe whose sequence is the same as or reverse complementary to the target probe binding region can hybridize with the target region or its amplification product. The reference region is obtained by replacing the target upstream primer binding region in the target region with a reference upstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 3' end) and / or replacing the target downstream primer binding region in the target region with a reference downstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 5' end).

2. A polynucleotide comprising N reference regions, where N is a natural number greater than or equal to 2, wherein, The nth reference region corresponds to the nth target region. The nth target region includes at least the nth target upstream primer binding region and the nth target downstream primer binding region, as well as the nth target probe binding region between them. The sequence of the nth target region is identical to or reverse complementary to the sequence of the nth target segment of the nth target gene of the nth target species. The nth target upstream primer whose sequence is identical to the nth target upstream primer binding region or whose 3' end contains the sequence of the nth target upstream primer binding region, and the nth target downstream primer whose sequence is reverse complementary to the nth target downstream primer binding region or whose 3' end contains the reverse complementary sequence of the nth target downstream primer binding region, together can be used with the nth target upstream primer binding region. The target region or a polynucleotide containing the nth target region is used as a template to amplify the nth target region. The nth target probe, whose sequence is the same as or reverse complementary to the nth target probe binding region, can hybridize with the nth target region or its amplification product. The nth reference region is obtained by replacing the nth target upstream primer binding region in the nth target region with a nth reference upstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 3' end) and / or replacing the nth target downstream primer binding region in the nth target region with a nth reference downstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 5' end). n is a natural number from 1 to N.

3. A polynucleotide according to claim 2, wherein: (1) The N target regions are pairwise distinct; 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. A group of polynucleotides contains N polynucleotides, where N is a natural number greater than 2. The nth polynucleotide contains an nth reference region, which corresponds to the nth target region. The nth target region includes at least an nth target upstream primer binding region and an nth target downstream primer binding region, as well as an nth target probe binding region between them. The sequence of the nth target region is identical to or reverse complementary to the sequence of the nth target segment of the nth target gene of the nth target species. The nth target upstream primer has a sequence identical to or contains the sequence of the nth target upstream primer binding region at its 3' end, and the nth target downstream primer has a sequence reverse complementary to or contains the reverse complementary sequence of the nth target downstream primer binding region at its 3' end. The device is capable of amplifying the nth target region using the nth target region or a polynucleotide containing the nth target region as a template. The nth target probe, whose sequence is the same as or reverse complementary to the nth target probe binding region, can hybridize with the nth target region or its amplification product. The nth reference region is obtained by replacing the nth target upstream primer binding region in the nth target region with a nth reference upstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 3' end) and / or replacing the nth target downstream primer binding region in the nth target region with a nth reference downstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 5' end), where n is a natural number from 1 to N.

5. The group of polynucleotides according to claim 4, wherein: (1) The N target regions are pairwise distinct; 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. A method for determining the presence of a target region in a test sample derived from a host species, comprising: Real-time quantitative PCR was performed simultaneously on the test tubes and the positive control tubes. The test tube contains a test sample with the 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 contains a positive reference material comprising 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. The positive reference polynucleotide includes a reference region corresponding to the target region. The target region includes at least a target upstream primer binding region, a target downstream primer binding region, and a target probe binding region between them. The sequence of the target region is identical to or reverse complementary to the sequence of the target segment of the target gene of the target species. The target upstream primer, whose sequence is identical to the target upstream primer binding region or whose 3' end contains the sequence of the target upstream primer binding region, and the target downstream primer, whose sequence is reverse complementary to the target downstream primer binding region or whose 3' end contains the reverse complementary sequence of the target downstream primer binding region, together can amplify the target region using the target region or a polynucleotide containing the target region as a template. The target probe, whose sequence is identical to or reverse complementary to the target probe binding region, can bind to the target probe. The target region or its amplification product is hybridized, wherein the reference region is obtained by replacing the target upstream primer binding region in the target region with a reference upstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 3' end) and / or replacing the target downstream primer binding region in the target region with a reference downstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 5' end). The reference upstream primer whose sequence is the same as the reference upstream primer binding region or whose 3' end contains the sequence of the reference upstream primer binding region, and the reference downstream primer whose sequence is reverse complementary to the reference downstream primer binding region or whose 3' end contains the reverse complementary sequence of the reference downstream primer binding region, together can amplify the reference region using the reference region or a polynucleotide containing the reference region as a template.

7. A method for determining the presence of N target regions in a test sample derived from a host species, where N is a natural number greater than or equal to 2, comprising: Real-time quantitative PCR was performed simultaneously on N test tubes and N positive control tubes. The nth test tube contains a test sample with the test nucleic acid as a template, the nth target upstream primer and the nth target downstream primer as primer pairs, and the nth target probe as a probe. The nth positive reference tube contains a positive reference material 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 includes an nth reference region, which corresponds to the nth target region. The nth target region includes at least an nth target upstream primer binding region and an nth target downstream primer binding region, as well as an nth target probe binding region between them. The sequence of the nth target region is identical to or reverse complementary to the sequence of the nth target segment of the nth target gene of the nth target species. An nth target upstream primer whose sequence is identical to or contains the sequence of the nth target upstream primer binding region at its 3' end, and an nth target downstream primer whose sequence is reverse complementary to or contains the reverse complementary sequence of the nth target downstream primer binding region at its 3' end, together can amplify the nth target region using the nth target region or a polynucleotide containing the nth target region as a template. An nth target probe whose sequence is identical to or reverse complementary to the nth target probe binding region can bind to the target gene. The nth target region or its amplification product is hybridized, wherein the nth reference region is obtained by replacing the nth target upstream primer binding region in the nth target region with a nth reference upstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 3' end) and / or replacing the nth target downstream primer binding region in the nth target region with a nth reference downstream primer binding region with a different sequence (e.g., at least 4 consecutive nucleotides different at the 5' end). The nth reference upstream primer whose sequence is the same as the nth reference upstream primer binding region or whose 3' end contains the sequence of the nth reference upstream primer binding region, and the nth reference downstream primer whose sequence is reverse complementary to the nth reference downstream primer binding region or whose 3' end contains the reverse complementary sequence of the nth reference downstream primer binding region, together can amplify the nth reference region using the nth reference region or a polynucleotide containing the nth reference region as a template, where n is a natural number from 1 to N.

8. The method according to claim 7, wherein: (1) N positive reference polynucleotides contain N reference regions, and the nth positive reference polynucleotide contains the nth reference region; or (2) N reference regions are contained in a single positive reference polynucleotide.

9. The method according to claim 7, wherein: (1) The N target regions are pairwise distinct; 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. A reagent kit comprising: (1) The polynucleotide according to claim 1; (2) A polynucleotide according to claim 2 or 3; or (3) A set of polynucleotides according to claim 4 or 5.

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