CN-121975954-A - Primer probe combination for identifying primary macula and gymnocypris spinosa and digital PCR (polymerase chain reaction) kit

CN121975954ACN 121975954 ACN121975954 ACN 121975954ACN-121975954-A

Abstract

The invention provides a primer probe combination and a digital PCR kit for identifying black plat original glabra and naked carp, wherein the primer probe combination is used for synchronously detecting black plat original glabra target DNA and naked carp target DNA in sample DNA on a micro-drop digital PCR platform and constructing a triple digital PCR detection reaction system, and the primer probe combination at least comprises a first primer pair and a first TaqMan probe aiming at specific sections of black plat original glabra target genes, a second primer pair and a second TaqMan probe aiming at specific sections of naked carp target genes, and an internal standard (IPC) primer pair and an internal standard probe for monitoring the process of nucleic acid extraction and amplification reaction. According to the invention, a triple detection reaction system of the original black-spot and naked carp targets and an internal standard in parallel is constructed on a microdroplet digital PCR platform, and different fluorescent channels are adopted for end point fluorescent interpretation, so that the detection result and the corresponding absolute copy number concentration of target DNA of two target species can be obtained in one detection of the same sample.

Inventors

ZHANG JIFENG
ZHAO NING
CAO PENGXI
Zeng Zhengkui
ZHAO YUN
HE SHENGLI

Assignees

西藏大学
四川杰莱美科技有限公司

Dates

Publication Date: 20260505
Application Date: 20260402

Claims (9)

1. The primer probe combination for identifying the black spot protoglyptosis and the gymnocypris przewalskii is characterized in that the primer probe combination is used for synchronously detecting the black spot protoglyptosis target DNA and the gymnocypris przewalskii target DNA in sample DNA on a micro-drop digital PCR platform, and constructing a triple digital PCR detection reaction system; The primer probe combination at least comprises: a first primer pair and a first TaqMan probe for a specific segment of a target gene of the primary Blueshoe's name; A second primer pair and a second TaqMan probe for a specific segment of the target gene of the gymnocypris; an internal standard (IPC) primer pair and an internal standard probe for monitoring the process of nucleic acid extraction and amplification reaction. The first TaqMan probe, the second TaqMan probe and the internal standard probe are respectively arranged to be capable of performing end point fluorescence interpretation in different fluorescence channels so as to realize parallel detection of targets of two target species and the internal standard.
2. The combination of the primers for identifying the tidea and the gymnocypris spinosa according to claim 1, wherein the first primer pair and the first TaqMan probe are designed for specific sections of the tidea mitochondrial ND5 gene, the second primer pair and the second TaqMan probe are designed for specific sections of the gymnocypris spinosa mitochondrial CYTB gene, the internal standard (IPC) primer pair and the internal standard probe for monitoring the nucleic acid extraction and amplification reaction process are designed for specific sections of the 16S rRNA gene, and the specific sections of the 16S rRNA gene are conserved sections of the fish mitochondrial 16S rRNA gene for process quality control monitoring of covering various fish samples.
3. The primer probe combination for identifying the black spot proto-macula and the naked carp according to claim 1, wherein the fluorescent reporter group of the first TaqMan probe is ROX, the fluorescent reporter group of the second TaqMan probe is FAM, the fluorescent reporter group of the internal standard probe is HEX, three-channel independent interpretation is formed in the digital PCR end point fluorescent reading stage, and the three-channel independent interpretation results respectively correspond to the black spot proto-macula target, the naked carp target and the internal standard are output.
4. The primer probe combination for identifying the black spot prochloraz and the gymnocypris spinosa according to claim 1, wherein the primer probe combination is subjected to qPCR primary screening and digital PCR verification, when non-target species or amphibious reptile genome DNA is used as a template, no positive endpoint fluorescent signal is generated by the black spot prochloraz channel and the gymnocypris spinosa channel, and the internal standard channel keeps normal amplification signals, so that false negatives caused by ' target undetected ' and ' reaction failure or inhibition can be distinguished.
5. A digital PCR kit for identifying macelian and gymnocypris, the kit comprising at least: (1) A specific primer probe mixture prepared by the primer probe combination of any one of claims 1-4, wherein the specific primer probe mixture comprises two sets of target primer pairs and probes and one set of internal standard primer pairs and probes, and is provided in a premixing mode for completing the construction of a triple system by one sample addition; (2) The digital PCR premix comprises a hot start DNA polymerase, dNTPs, mgCl 2 and a buffer system; (3) A positive quality control comprising recombinant plasmid DNA or synthetic nucleic acid fragments of a primary jetty sequence and a target sequence of a gymnocypris przewalskii; (4) A negative quality control, wherein the negative quality control is nuclease-free water; The kit is configured into a digital PCR detection flow suitable for microdroplet separation, PCR amplification and end point fluorescence reading, and is used for constructing a detection configuration of a single or multiple digital PCR reaction system.
6. The digital PCR kit for identifying the prochloraz jetlag and the gymnocypris spinosa according to claim 5, wherein the internal standard primer pair and the internal standard probe are used for carrying out quality control monitoring on the reaction process and judging that the sample DNA extraction fails, the reaction is invalid or the PCR inhibition exists when the expected positive endpoint fluorescence judgment result does not appear in the internal standard channel; wherein the "expected positive endpoint fluorescence interpretation" comprises that the internal standard channel forms a distinguishable set of positive microreaction units upon endpoint fluorescence reading.
7. A digital PCR detection method for identifying maciteus maculatus and gymnocypris spinosa, characterized in that the digital PCR kit according to claim 5 or 6 is used for detecting sample DNA, the method comprising the steps of: s1, obtaining a fish tissue sample or a water body environment sample and extracting DNA; S2, mixing the extracted DNA with a specific primer probe mixed solution and a digital PCR premix to form a reaction system, and setting a positive quality control product and a negative quality control product; S3, carrying out microdroplet separation on the reaction system to form a plurality of independent micro-reaction units, and carrying out PCR amplification; S4, performing end-point fluorescence reading on the micro-reaction units after amplification is finished, respectively judging positive or negative states of the black spot original target, the nude carp target and the internal standard based on different fluorescence channels, and counting the number of positive micro-reaction units and the total micro-reaction units of each channel to form quantitative calculation input.
8. The digital PCR detection method for identifying the black spot prochloraz and the gymnocypris przewalskii according to claim 7 is characterized in that after finishing the end point fluorescence interpretation, the proportion of positive micro-reaction units is converted based on a poisson statistical model so as to calculate the absolute copy number concentration of the black spot prochloraz target DNA and the gymnocypris przewalskii target DNA; Wherein the ratio of positive micro-reaction units is determined by the ratio of the number of positive micro-reaction units to the total number of micro-reaction units.
9. The digital PCR detection method for identifying the prochloraz jerina and the gymnocypris przewalskii according to claim 7 is characterized in that the digital PCR detection method according to claim 7 or 8 is adopted to detect the extracted environmental DNA in a water body sample for judging the existence and relative abundance change of the prochloraz and the gymnocypris przewalskii in a target water area, and the water body sample can be subjected to filtration or centrifugation enrichment treatment before DNA extraction to obtain the environmental DNA.

Description

Primer probe combination for identifying primary macula and gymnocypris spinosa and digital PCR (polymerase chain reaction) kit Technical Field The invention relates to the crossing fields of molecular biology, environmental ecology and species protection monitoring technology, in particular to a primer probe combination and a digital PCR kit for identifying original red-blue and nux-blue carp. Background The plateau in the Tibet area is an important water source conservation area and a concentrated biodiversity distribution area, and various specific fish resources are distributed in the water area ecological system. The black plagion (Glyptosternum maculatum) and the gymnocypris spinosa (Oxygymnocypris stewartii) are fish special for the Qinghai-Tibet plateau, belong to important protection objects in the country and the place respectively, and play an important role in maintaining the ecological structural stability of the high-raw water domain. The population quantity of the fishes is in a descending trend under the influence of the factors such as hydrologic condition change, habitat damage, human activities and the like, and long-term and fine monitoring and evaluation work is needed to be carried out. The existing fish resource investigation mainly depends on field fishing and morphological identification, has the problems of strong invasiveness, low investigation efficiency, insufficient capability of detecting rare or hidden species and the like, and is difficult to meet the continuous monitoring requirement under the condition of complex water areas on a plateau. At present, a common real-time fluorescent quantitative PCR (qPCR) method in eDNA detection relies on a standard curve to carry out relative quantification, is easily influenced by PCR inhibitors in complex environment samples, has limited quantitative stability and low copy detection capability, and has higher construction difficulty of a multi-target synchronous detection system; The digital PCR (dPCR) can realize absolute quantification of target DNA through micro-reaction unit separation and end point signal statistical analysis, has stronger inhibitor tolerance, is suitable for low abundance target detection, and is nevertheless lack of synchronous, specific and absolute quantitative detection for black spot original and gymnocypris przewalskii aiming at eDNA monitoring of endangered fishes in Qinghai-Tibet plateau; For this purpose, a primer probe combination and a digital PCR kit for identifying the black-spotted primordial-red and the naked carp are proposed. Disclosure of Invention In view of the above, the invention provides a primer probe combination for identifying the prochloraz jetlag and the gymnocypris, so as to solve or alleviate the technical problems existing in the prior art, and at least provide a beneficial choice. The technical scheme of the invention is realized by combining a primer probe for identifying the original macelian and the naked cyprinus carpio, wherein the primer probe combination is used for synchronously detecting the original macelian target DNA and the naked cyprinus carpio target DNA in sample DNA on a micro-drop type digital PCR platform and constructing a triple digital PCR detection reaction system; The primer probe combination at least comprises: a first primer pair and a first TaqMan probe for a specific segment of a target gene of the primary Blueshoe's name; A second primer pair and a second TaqMan probe for a specific segment of the target gene of the gymnocypris; An internal standard (IPC) primer pair and an internal standard probe for monitoring the process of nucleic acid extraction and amplification reaction; The first TaqMan probe, the second TaqMan probe and the internal standard probe are respectively arranged to be capable of performing end point fluorescence interpretation in different fluorescence channels so as to realize parallel detection of targets of two target species and the internal standard. Further preferably, the first primer pair and the first TaqMan probe are designed for a specific segment of the black spot primary-horn mitochondrial ND5 gene, the second primer pair and the second TaqMan probe are designed for a specific segment of the gymnocypris spinosa mitochondrial CYTB gene, the internal standard (IPC) primer pair and the internal standard probe for monitoring the process of the nucleic acid extraction and amplification reaction are designed for a specific segment of the 16S rRNA gene, and the specific segment of the 16S rRNA gene is a conserved segment of the fish mitochondrial 16S rRNA gene and is used for process quality control monitoring of covering various fish samples. Further preferably, the fluorescent reporter group of the first TaqMan probe is ROX, the fluorescent reporter group of the second TaqMan probe is FAM, the fluorescent reporter group of the internal standard probe is HEX, three channels are formed for independent interpretation in the digit