CN113403420A - Primer probe combination for detecting yersinia sporogenes and drug-resistant mutation thereof and application of primer probe combination - Google Patents
Primer probe combination for detecting yersinia sporogenes and drug-resistant mutation thereof and application of primer probe combination Download PDFInfo
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Abstract
The invention provides a primer probe combination for detecting yersinia pneumocystis and drug-resistant mutation thereof and application thereof, wherein the primer probe combination for detecting the yersinia pneumocystis and the drug-resistant mutation thereof comprises a primer pair and a probe for amplifying and detecting dihydrofolate synthetase genes; the primer pair for amplifying the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 1-2, and the probe for detecting the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 3. The invention also provides a kit for detecting the pneumocystis yezoensis and the drug-resistant mutation thereof, a use method thereof for non-disease diagnosis and/or treatment, and a device for detecting the pneumocystis yezoensis and the drug-resistant mutation thereof. The primer probe combination has good specificity and high sensitivity, and the kit has reasonable design, convenient use and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer probe combination for detecting yersinia pneumocystis and drug-resistant mutation thereof and application thereof.
Background
Pneumocystis (Pneumocystis) is a unicellular organism that has long been classified as a protozoan, known as Pneumocystis carinii. In 1942, jirovic et al isolated the pathogen from lung tissue of patients with pneumonia for the first time. In 1988, it was confirmed that it belongs to fungi, more commonly referred to as pneumocystis, by sequence analysis of its ribosomal small subunit rRNA. According to the related studies, it is found that the Pneumocystis bacteria infected human and animal are different, and the disease caused by the Pneumocystis is called Pneumocystis disease (Pneumocysis) which is named as Pneumocystis jiirochachi.
The yersinia Pneumocystis is an opportunistic pathogen, which can cause serious yersinia Pneumocystis Pneumonia (PCP), and is often seen in people with low Immune function such as AIDS patients and other people with immunodeficiency and other Immune function defects. According to statistics, the probability of the AIDS patients with the combined infection of the yersinia sporophyte pneumonia is up to 70-80%, and if the AIDS patients are not treated, the mortality rate is up to 100%. In recent years, with the widespread use of broad-spectrum antibiotics, corticosteroids and immunosuppressants, and the increase of patients with diabetes and aids, etc., good conditions are provided for the propagation and pathogenesis of conditional pathogenic fungi, and the incidence of the pneumocystis yezoensis pneumonia infection tends to increase significantly.
The diagnosis of the pneumocystis Yersiniae pneumonia infection faces many difficulties, PCP lacks specific clinical symptoms, and the pathogenic detection is the only evidence for the definite diagnosis of PCP, but the pneumocystis Yersiniae can not be cultured in vitro, and generally adopts a staining method for diagnosis, but the used sample is generally lung tissue or alveolar lavage fluid (BALF), so that a material for creating the sample is needed, and the application of the method is limited to a great extent; the serological detection has high false positive rate and low specificity; there are also methods for detection by molecular biology means, but it is only possible to detect if the infection is by pneumocystis yeri, and it is not possible to distinguish pneumocystis yeri and its drug-resistant mutation, and the detection method is complicated, and the detection cost is high by using a plurality of pairs of primer probes.
Therefore, it is a urgent need to provide a method for detecting Yersinia pneumocystis, which can detect whether a sample is infected with Yersinia pneumocystis and distinguish the drug-resistant mutation type.
Disclosure of Invention
Aiming at the defects and the actual requirements of the prior art, the invention provides a primer probe combination for detecting the yersinia sporogenes and the drug-resistant mutation thereof and the application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer probe combination for detecting yersinia pneumocystis and drug-resistant mutation thereof, wherein the primer probe combination for detecting yersinia pneumocystis and drug-resistant mutation thereof comprises a primer pair and a probe for amplifying and detecting dihydrofolate synthetase genes;
the primer pair for amplifying the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 1-2, and the probe for detecting the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 3.
SEQ ID No.1：ATTAATGGATGTGGAGAATTT；
SEQ ID No.2：TTTTATAGCAGGAATAACTCG；
SEQ ID No.3：TGGGCAGTCTGCACGGTCTGGTT。
In the invention, only one pair of primer probes is used, the detection can be carried out on the yersinia sporogenes in the sample, and the wild type W and three mutations, namely the double-site mutation M, the single-site mutation MG (T55A) and the single-site mutation MT (P57S) are all distinguished, so that the time consumption is extremely short, and the detection efficiency is high; the dihydrofolate synthase (DHPS) is selected as a target gene, so that the sensitivity and the specificity are high, the operation is simple, and the detection cost is low.
In the invention, the wild type sequence of the yersinia pneumocystis is shown in SEQ ID No.7, and the mutant type sequence is shown in SEQ ID No. 8.
SEQ ID No.7：
agttctgaattttataaagcgcctacacatattatggccattttaaatcttactcctgattcttttttcgatgggggtgttcattcatatgattctatattaatggatgtggagaattttataaatgcaggggcgacgataattgatattggtgggcagtctacacggcctggttcacatgttgtttctatagaggaagagatttctcgagttattcctgctataaaatatctcttaaaagtatatcctgatattttagtaagtgtagatacttttcgttctgaggttgcagaacaagcaattaaggctggtgctagtcttgttaatgatataagtgggggaaggtatgatccaaaaatgcttaatgtggttgccaagttgaaagttccaatatgtataatgcatatgagaggtgattttttaactatggacaatttaactgattatggtaccgatattataaaacaaattactaaagaattagaagaattgcttgtttttgctgaaagttcgggtatttttaggtggaatattattttagatcctgggttaggatttgctaaaacttcctatcaaaatatagaattgttaagaagatttaatgaattaaaatctcagcattgctttaatggtttgccttggttgcttggtccaagtcgcaaaagatttacagggtgtcttacaggtgatgttatgccaaaagataggatttggggcactgctgcttcggttgccgcatctgttttaggaggctgtgatattatacgggttcatgatgtttatgaaatgtataaagtttcaagaactttggatgctatttggaaagaaatttattaa。
SEQ ID No.8：
agttctgaattttataaagcgcctacacatattatggccattttaaatcttactcctgattcttttttcgatgggggtgttcattcatatgattctatattaatggatgtggagaattttataaatgcaggggcgacgataattgatattggtgggcagtctgcacggtctggttcacatgttgtttctatagaggaagagatttctcgagttattcctgctataaaatatctcttaaaagtatatcctgatattttagtaagtgtagatacttttcgttctgaggttgcagaacaagcaattaaggctggtgctagtcttgttaatgatataagtgggggaaggtatgatccaaaaatgcttaatgtggttgccaagttgaaagttccaatatgtataatgcatatgagaggtgattttttaactatggacaatttaactgattatggtaccgatattataaaacaaattactaaagaattagaagaattgcttgtttttgctgaaagttcgggtatttttaggtggaatattattttagatcctgggttaggatttgctaaaacttcctatcaaaatatagaattgttaagaagatttaatgaattaaaatctcagcattgctttaatggtttgccttggttgcttggtccaagtcgcaaaagatttacagggtgtcttacaggtgatgttatgccaaaagataggatttggggcactgctgcttcggttgccgcatctgttttaggaggctgtgatattatacgggttcatgatgtttatgaaatgtataaagtttcaagaactttggatgctatttggaaagaaatttattaa。
Preferably, the primer probe combination for detecting the yersinia sporogenes and the drug-resistant mutation thereof further comprises a primer pair and a probe for amplifying and detecting an internal standard gene.
Preferably, the primer pair for amplifying the internal standard gene comprises the nucleotide sequence shown in SEQ ID No. 4-5, and the probe for detecting the internal standard gene comprises the nucleotide sequence shown in SEQ ID No. 6.
SEQ ID No.4：CTGAGTCGGGATGTGCTA；
SEQ ID No.5：ACCAGTATCTACCGCCAAA；
SEQ ID No.6：ACGGAACCATTCGCTCGCCAATCG。
Preferably, the probe carries a fluorescent group and a quencher group.
Preferably, the fluorophore comprises any one of ALEX-350, Alexa Fluor 488, CY3, FAM, VIC, TET, CALGold540, JOE, HEX, CALFluorOrange560, TAMRA, CALFluorRed590, ROX, CALFluorRed610, TexasRed, CALFluorRed635, Quasar670, CY5, CY5.5, LC RED640, or Quasar 705.
Preferably, the quenching group comprises any one of TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 or Eclipse.
In the present invention, the 5 'end of each probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. In the PCR amplification process, when the probe is complete, the fluorescence energy emitted by the fluorescent group is absorbed by the quenching group, and the signal cannot be detected by an instrument. When the primer is extended, the probe bound to the template is cleaved by Taq enzyme (5 '→ 3' exonuclease activity), the fluorescent group is away from the quencher, and its energy cannot be absorbed, i.e., a fluorescent signal is generated. After PCR amplification is finished, the system is subjected to melting curve analysis, and according to different Tm values, the whole differentiation of the yersinia sporogenes and the drug-resistant mutation thereof can be realized in a single channel.
In a second aspect, the invention provides a kit for detecting pneumocystis yezoensis and drug-resistant mutation thereof, and the kit for detecting pneumocystis yezoensis and drug-resistant mutation thereof comprises the primer-probe combination for detecting pneumocystis yezoensis and drug-resistant mutation thereof in the first aspect.
Preferably, the kit for detecting yersinia sporogenes and drug-resistant mutation thereof comprises PCR detection liquid containing a primer probe combination for detecting the yersinia sporogenes and the drug-resistant mutation thereof, the adding amount ratio of the upstream primer to the downstream primer in the PCR detection liquid is 1 (48-52), for example, 1:48, 1:49, 1:50, 1:51 or 1:52, and the like, and other specific point values in the numerical range can be selected, so that the detailed description is omitted.
In the present invention, amplification is performed by asymmetric PCR, and a large amount of single-stranded DNA (ssDNA) is generated after PCR amplification using an unequal number of a pair of primers. The pair of primers is generally referred to as a limiting primer and a non-limiting primer, the limiting primer being a low concentration primer and the non-limiting primer being a high concentration primer. In the first 10-15 cycles of PCR reaction, the amplification product is mainly double-stranded DNA, and after the restriction primers are consumed, a large amount of single-stranded DNA is generated by the non-restriction primers. The proportion of the two primers has important influence on the result of asymmetric PCR reaction, and the invention controls the adding amount proportion of the upstream primer and the downstream primer to be 1 (48-52), thereby achieving the best amplification effect and ensuring more accurate detection result.
Preferably, the kit for detecting the yersinia sporogenes and the drug-resistant mutation thereof further comprises PCR mixed liquor, internal standard quality control, positive quality control and negative quality control.
Preferably, the PCR mixture comprises DNA polymerase, dNTPs and Mg2+And a buffer.
Preferably, the internal standard quality control comprises a plasmid containing a detection site sequence of an internal standard gene.
Preferably, the positive quality control comprises a plasmid containing the sequence of the detection site of pneumocystis Yersinia.
Preferably, the negative quality control comprises water.
In a third aspect, the present invention provides a method for using the kit for detecting yersinia sporogenes and drug-resistant mutations thereof in the second aspect for the purpose of non-disease diagnosis and/or treatment, comprising the steps of:
extracting nucleic acid of the sample, performing fluorescence quantitative PCR amplification, analyzing a melting curve, and judging the pneumocystis yedoensis and the drug-resistant mutation condition thereof in the sample.
In a fourth aspect, the present invention provides an apparatus for detecting pneumocystis yezoensis and drug-resistant mutations thereof, comprising:
a nucleic acid extraction module: nucleic acids for extracting a sample;
an amplification module: performing fluorescent quantitative PCR amplification by using the obtained nucleic acid as a template;
an analysis module: and analyzing the melting curve of the amplification reaction, and judging the pneumocystis yezoensis and the drug-resistant mutation condition of the pneumocystis yezoensis in the sample.
In a fifth aspect, the present invention provides the use of any one or a combination of at least two of the primer-probe combination for detecting pneumocystis yezoensis and drug-resistant mutation thereof described in the first aspect, the kit for detecting pneumocystis yezoensis and drug-resistant mutation thereof described in the second aspect, the method for using the kit for detecting pneumocystis yezoensis and drug-resistant mutation thereof described in the third aspect for non-disease diagnosis and/or treatment, or the device for detecting pneumocystis yezoensis and drug-resistant mutation thereof described in the fourth aspect, for detecting drug-resistant mutation of pneumocystis yezoensis.
Compared with the prior art, the invention has the following beneficial effects:
(1) the primer probe combination for detecting the yersinia sporogenes and the drug-resistant mutation thereof has good specificity and sensitivity, and is matched with a corresponding detection reagent, the kit for detecting the yersinia sporogenes and the drug-resistant mutation thereof is convenient to use and simple to operate, and can monitor the whole process of detection reaction by being matched with an internal standard gene primer probe, internal standard quality control, positive quality control and negative quality control, so that the result is more real and credible;
(2) the invention can realize the rapid diagnosis of the infection of the yersinia sporogenes, can detect the drug resistance of sulfonamides in the same fluorescence channel, can carry out early, rapid and accurate screening diagnosis on whether the sample is infected with the yersinia sporogenes and whether the sample has the drug resistance, does not need culture experiments, has short time consumption, can obtain accurate results within 2h, has important significance for prognosis, is a new direction for the infection diagnosis of the yersinia sporogenes and has important practical significance.
Drawings
FIG. 1 is a photograph of a melting curve in example 3;
FIG. 2 is a graph showing a melting curve in example 4;
FIG. 3 is a graph showing a melting curve in example 5;
FIG. 4 is a photograph showing a melting curve in example 6.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Materials:
the nucleic acid extraction Kit is purchased from Roche's High Pure PCR Template Preparation Kit, Inc. (Shanghai);
the sample is from China general microbiological culture Collection center;
PCR mix was purchased from roche diagnostics products (shanghai) ltd;
the internal standard quality control plasmid and the positive quality control plasmid were purchased from Biotechnology engineering (Shanghai) GmbH.
Example 1
The embodiment provides a primer probe combination for detecting yersinia pneumocystis and drug-resistant mutation thereof, which comprises a primer pair and a probe for amplifying and detecting dihydrofolate synthetase genes;
the primer pair for amplifying the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 1-2, and the probe for detecting the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 3.
SEQ ID No.1：ATTAATGGATGTGGAGAATTT；
SEQ ID No.2：TTTTATAGCAGGAATAACTCG；
SEQ ID No.3：TGGGCAGTCTGCACGGTCTGGTT。
The primer probe combination for detecting the yersinia sporogenes and the drug-resistant mutation thereof also comprises a primer pair and a probe for amplifying and detecting the internal standard gene.
The primer pair for amplifying the internal standard gene comprises nucleotide sequences shown in SEQ ID No. 4-5, and the probe for detecting the internal standard gene comprises nucleotide sequences shown in SEQ ID No. 6.
SEQ ID No.4：CTGAGTCGGGATGTGCTA；
SEQ ID No.5：ACCAGTATCTACCGCCAAA；
SEQ ID No.6：ACGGAACCATTCGCTCGCCAATCG。
The 5 'end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. The fluorescent group of the probe for detecting the yersinia pneumocystis and the drug-resistant mutation of the yersinia pneumocystis is CY5, and the quenching group is BHQ 2; the fluorescent group of the probe for detecting the internal standard gene is FAM, and the quenching group is BHQ 1.
In the invention, the infection condition of the yersinia sporogenes of a sample and whether the infection condition has drug-resistant mutation can be detected by a pair of primers and probes; the detection primers and probes of the internal standard genes are arranged, so that the detection process can be monitored in real time, and the detection result is more real and accurate.
Example 2
The present embodiment provides a kit for detecting pneumocystis yezoensis and drug-resistant mutations thereof, which includes the primer-probe combination for detecting pneumocystis yezoensis and drug-resistant mutations thereof in embodiment 1.
The kit for detecting the pneumocystis yezoensis and the drug-resistant mutation thereof comprises PCR detection liquid containing a primer probe combination for detecting the pneumocystis yezoensis and the drug-resistant mutation thereof, and the adding amount ratio of an upstream primer to a downstream primer in the PCR detection liquid is 1: 50.
The kit for detecting the yersinia sporogenes and the drug-resistant mutation thereof also comprises PCR mixed liquor, internal standard quality control, positive quality control and negative quality control;
the PCR mixed solution comprises DNA polymerase, dNTPs and Mg2+And a buffer;
the internal standard quality control comprises a plasmid containing a detection site sequence of an internal standard gene;
the positive quality control comprises a plasmid containing a detection site sequence of pneumocystis yezoensis;
the negative quality control comprises water.
The kit for detecting the yersinia sporogenes and the drug-resistant mutation thereof has scientific and reasonable design, can monitor the detection process by internal standard quality control, positive quality control and negative quality control, and has the advantages of more rigorous detection experiment, simple operation and convenient use.
Example 3
In this embodiment, the detection of the sample using the kit for detecting yersinia sporogenes and the drug-resistant mutation thereof prepared in example 2 comprises the following specific steps:
(1) extracting nucleic acid of a sample
The sample nucleic acid is extracted with reference to the instructions in the nucleic acid extraction kit.
(2) Fluorescent quantitative PCR amplification
The reaction system for fluorescent quantitative PCR amplification is as follows:
the reaction procedure for fluorescent quantitative PCR amplification was as follows:
pre-denaturation: at 95 ℃ for 10 min;
and (3) circulating amplification: 95 ℃ for 10 s; at 55 ℃ for 40 s; circulating for 45 times;
forming a melting curve: at 95 ℃ for 1 min; at 40 ℃ for 2 min; continuously collecting at 95 ℃ at a speed of 0.01 ℃/s;
and (3) cooling: 37 ℃ for 10 s.
(3) Analysis of
And analyzing the melting curve, and judging the pneumocystis yedoensis and the drug-resistant mutation condition thereof in the sample.
The criteria for the judgment are shown in Table 1.
TABLE 1
Example 4
The difference from example 3 is only that the ratio of the upstream primer to the downstream primer in the PCR detection solution in this example is 1:100, and the rest of the samples, materials and detection method are the same as example 3.
Example 5
The difference from example 3 is only that the ratio of the upstream primer to the downstream primer in the PCR detection solution in this example is 1:150, and the rest of the samples, materials and detection method are the same as example 3.
Example 6
The difference from example 3 is only that the ratio of the upstream primer to the downstream primer in the PCR detection solution in this example is 1:200, and the rest of the samples, materials and detection method are the same as example 3.
The melting curves of example 3 are shown in fig. 1, example 4 in fig. 2, example 5 in fig. 3, and example 6 in fig. 4.
As can be seen from the figure, the method of using fluorescent probe combined with melting curve in example 3 can clearly distinguish the infection of Yersinia pneumocystis and drug-resistant mutation thereof in the same channel, wherein the Tm value of the wild type W is 66.0-67.0; the Tm value of the single-site mutation MT (P57S) is 68.0-70; the Tm value of the single-site mutation MG (T55A) is 71.5-73.0; the Tm value of the double site mutation M is 74.0-75.0, and all fluorescence values are very similar. In example 4, the single-site mutation MG (T55A) and the double-site mutation M showed relatively low fluorescence values, and the Tm values were close to each other, so that they were not easily distinguishable. In example 5, the fluorescence values of the single-site mutation MT (P57S) and the double-site mutation M were low, and the Tm values of the single-site mutation MT (P57S) and the single-site mutation MG (T55A) were close to each other and could not be distinguished easily. In example 6, the fluorescence values of the single-site mutation MT (P57S) and the double-site mutation M were too low, and the Tm values of the single-site mutation MT (P57S), the single-site mutation MG (T55A) and the double-site mutation M were similar and could not be easily distinguished. In summary, in example 3, only the ratio of the addition amounts of the upstream primer and the downstream primer is controlled to be 1 (48-52), the amplification efficiencies of different primer pairs are close, the Tm value difference is large, the discrimination is easy, and the detection result is more accurate.
In conclusion, the primer probe for detecting the yersinia sporogenes and the drug-resistant mutation thereof has good combination specificity and high sensitivity; the kit for detecting the yersinia sporogenes and the drug-resistant mutation thereof is reasonable in design and simple and convenient to operate, can be matched with a corresponding detection method, can determine whether the yersinia sporogenes is infected by alveolar lavage fluid serving as a sample through one-time detection, can distinguish the drug-resistant mutation by using asymmetric PCR in a fluorescence channel, and is provided with an internal standard system to detect the whole detection process, so that the result is more accurate and reliable; compared with the traditional diagnosis method, the method has lower cost and simpler operation.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Dana (Tianjin) Biotechnology Ltd
<120> primer probe combination for detecting yersinia pneumocystis and drug-resistant mutation thereof and application thereof
<130> 2021
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
attaatggat gtggagaatt t 21
<210> 2
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<212> DNA
<213> Artificial sequence
<400> 2
ttttatagca ggaataactc g 21
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
tgggcagtct gcacggtctg gtt 23
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<211> 18
<212> DNA
<213> Artificial sequence
<400> 4
ctgagtcggg atgtgcta 18
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence
<400> 5
accagtatct accgccaaa 19
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence
<400> 6
acggaaccat tcgctcgcca atcg 24
<210> 7
<211> 837
<212> DNA
<213> Pneumocystis yeri
<400> 7
agttctgaat tttataaagc gcctacacat attatggcca ttttaaatct tactcctgat 60
tcttttttcg atgggggtgt tcattcatat gattctatat taatggatgt ggagaatttt 120
ataaatgcag gggcgacgat aattgatatt ggtgggcagt ctacacggcc tggttcacat 180
gttgtttcta tagaggaaga gatttctcga gttattcctg ctataaaata tctcttaaaa 240
gtatatcctg atattttagt aagtgtagat acttttcgtt ctgaggttgc agaacaagca 300
attaaggctg gtgctagtct tgttaatgat ataagtgggg gaaggtatga tccaaaaatg 360
cttaatgtgg ttgccaagtt gaaagttcca atatgtataa tgcatatgag aggtgatttt 420
ttaactatgg acaatttaac tgattatggt accgatatta taaaacaaat tactaaagaa 480
ttagaagaat tgcttgtttt tgctgaaagt tcgggtattt ttaggtggaa tattatttta 540
gatcctgggt taggatttgc taaaacttcc tatcaaaata tagaattgtt aagaagattt 600
aatgaattaa aatctcagca ttgctttaat ggtttgcctt ggttgcttgg tccaagtcgc 660
aaaagattta cagggtgtct tacaggtgat gttatgccaa aagataggat ttggggcact 720
gctgcttcgg ttgccgcatc tgttttagga ggctgtgata ttatacgggt tcatgatgtt 780
tatgaaatgt ataaagtttc aagaactttg gatgctattt ggaaagaaat ttattaa 837
<210> 8
<211> 837
<212> DNA
<213> Pneumocystis yeri
<400> 8
agttctgaat tttataaagc gcctacacat attatggcca ttttaaatct tactcctgat 60
tcttttttcg atgggggtgt tcattcatat gattctatat taatggatgt ggagaatttt 120
ataaatgcag gggcgacgat aattgatatt ggtgggcagt ctgcacggtc tggttcacat 180
gttgtttcta tagaggaaga gatttctcga gttattcctg ctataaaata tctcttaaaa 240
gtatatcctg atattttagt aagtgtagat acttttcgtt ctgaggttgc agaacaagca 300
attaaggctg gtgctagtct tgttaatgat ataagtgggg gaaggtatga tccaaaaatg 360
cttaatgtgg ttgccaagtt gaaagttcca atatgtataa tgcatatgag aggtgatttt 420
ttaactatgg acaatttaac tgattatggt accgatatta taaaacaaat tactaaagaa 480
ttagaagaat tgcttgtttt tgctgaaagt tcgggtattt ttaggtggaa tattatttta 540
gatcctgggt taggatttgc taaaacttcc tatcaaaata tagaattgtt aagaagattt 600
aatgaattaa aatctcagca ttgctttaat ggtttgcctt ggttgcttgg tccaagtcgc 660
aaaagattta cagggtgtct tacaggtgat gttatgccaa aagataggat ttggggcact 720
gctgcttcgg ttgccgcatc tgttttagga ggctgtgata ttatacgggt tcatgatgtt 780
tatgaaatgt ataaagtttc aagaactttg gatgctattt ggaaagaaat ttattaa 837
Claims (10)
1. The primer probe combination for detecting the yersinia sporophyte and the drug-resistant mutation thereof is characterized by comprising a primer pair and a probe for amplifying and detecting a dihydrofolate synthetase gene;
the primer pair for amplifying the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 1-2, and the probe for detecting the dihydrofolate synthetase gene comprises a nucleotide sequence shown in SEQ ID No. 3.
2. The primer-probe combination for detecting Yersinia pneumocystis and drug-resistant mutation thereof of claim 1, wherein said primer-probe combination for detecting Yersinia pneumocystis and drug-resistant mutation thereof further comprises a primer pair and a probe for amplifying and detecting an internal standard gene.
3. The primer probe combination for detecting Yersinia pneumocystis and drug-resistant mutation thereof according to claim 2, wherein the primer pair for amplifying the internal standard gene comprises the nucleotide sequence shown in SEQ ID No. 4-5, and the probe for detecting the internal standard gene comprises the nucleotide sequence shown in SEQ ID No. 6.
4. The primer-probe combination for detecting Yersinia pneumocystis and drug-resistant mutation thereof according to any one of claims 1 to 3, wherein the probe carries a fluorescent group and a quenching group;
preferably, the fluorophore comprises any one of ALEX-350, Alexa Fluor 488, CY3, FAM, VIC, TET, CALGold540, JOE, HEX, CALFluorOrange560, TAMRA, CALFluorRed590, ROX, CALFluorRed610, TexasRed, CALFluorRed635, Quasar670, CY5, CY5.5, LC RED640, or Quasar 705;
preferably, the quenching group comprises any one of TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 or Eclipse.
5. A kit for detecting pneumocystis yezoensis and drug-resistant mutation thereof, wherein the kit for detecting pneumocystis yezoensis and drug-resistant mutation thereof comprises the primer-probe combination for detecting pneumocystis yezoensis and drug-resistant mutation thereof according to any one of claims 1 to 4.
6. The kit for detecting Yersinia pneumocystis and drug-resistant mutation thereof according to claim 5, wherein said kit for detecting Yersinia pneumocystis and drug-resistant mutation thereof comprises a PCR detection solution containing said primer-probe combination for detecting Yersinia pneumocystis and drug-resistant mutation thereof, and the ratio of the addition amount of the upstream primer and the downstream primer in said PCR detection solution is 1 (48-52).
7. The kit for detecting Yersinia pneumocystis and drug-resistant mutation thereof according to claim 5 or 6, wherein the kit for detecting Yersinia pneumocystis and drug-resistant mutation thereof further comprises PCR mixture, internal standard quality control, positive quality control and negative quality control;
preferably, the PCR mixture comprises DNA polymerase, dNTPs and Mg2+And a buffer;
preferably, the internal standard quality control comprises a plasmid containing a detection site sequence of an internal standard gene;
preferably, the positive quality control comprises a plasmid containing a detection site sequence of pneumocystis yezoensis;
preferably, the negative quality control comprises water.
8. The use of the kit for detecting Yersinia pneumonocardia and its drug-resistant mutation according to any one of claims 5 to 7 for non-disease diagnosis and/or treatment purposes, comprising the steps of:
extracting nucleic acid of the sample, performing fluorescence quantitative PCR amplification, analyzing a melting curve, and judging the pneumocystis yedoensis and the drug-resistant mutation condition thereof in the sample.
9. An apparatus for detecting pneumocystis yezoensis and drug-resistant mutations thereof, comprising:
a nucleic acid extraction module: nucleic acids for extracting a sample;
an amplification module: performing fluorescent quantitative PCR amplification by using the obtained nucleic acid as a template;
an analysis module: and analyzing the melting curve of the amplification reaction, and judging the pneumocystis yezoensis and the drug-resistant mutation condition of the pneumocystis yezoensis in the sample.
10. The use of any one or a combination of at least two of the primer-probe combination for detecting pneumocystis yedoensis and drug-resistant mutation thereof according to any one of claims 1 to 4, the kit for detecting pneumocystis yedoensis and drug-resistant mutation thereof according to any one of claims 5 to 7, the method for using the kit for detecting pneumocystis yedoensis and drug-resistant mutation thereof according to claim 8 for the purpose of non-disease diagnosis and/or treatment, or the device for detecting pneumocystis yedoensis and drug-resistant mutation thereof according to claim 9 for the detection of drug-resistant mutation of pneumocystis yedoensis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113718053A (en) * | 2021-09-30 | 2021-11-30 | 北京大学第一医院 | Probe and primer pair for detecting yersinia sporogenes, detection method and application |
| CN117965800A (en) * | 2024-03-29 | 2024-05-03 | 南京诺因生物科技有限公司 | Compositions and kits for QPCR-based detection of Aspergillus fumigatus, yersinia pneumoconica and Cryptococcus neoformans |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142789A (en) * | 2013-03-15 | 2015-12-09 | 纳诺拜希姆公司 | Systems and methods for mobile device analysis of nucleic acids and proteins |
| CN112063733A (en) * | 2020-09-21 | 2020-12-11 | 上海捷诺生物科技有限公司 | Kit, reaction system and method for detecting pneumocystis and drug resistance thereof |
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2021
- 2021-08-05 CN CN202110895392.2A patent/CN113403420A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142789A (en) * | 2013-03-15 | 2015-12-09 | 纳诺拜希姆公司 | Systems and methods for mobile device analysis of nucleic acids and proteins |
| CN112063733A (en) * | 2020-09-21 | 2020-12-11 | 上海捷诺生物科技有限公司 | Kit, reaction system and method for detecting pneumocystis and drug resistance thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113718053A (en) * | 2021-09-30 | 2021-11-30 | 北京大学第一医院 | Probe and primer pair for detecting yersinia sporogenes, detection method and application |
| CN117965800A (en) * | 2024-03-29 | 2024-05-03 | 南京诺因生物科技有限公司 | Compositions and kits for QPCR-based detection of Aspergillus fumigatus, yersinia pneumoconica and Cryptococcus neoformans |
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Application publication date: 20210917 |