1. The primer probe combination for mucor typing detection is characterized by comprising any one or a combination of at least two of a primer pair and a probe for amplifying and detecting rhizopus, a primer pair and a probe for amplifying and detecting mucor, a primer pair and a probe for amplifying and detecting absidia, a primer pair and a probe for amplifying and detecting rhizopus and a primer pair and a probe for amplifying and detecting kummer;

the detection sites of the primer probe combination for mucor typing detection are positioned in the 28S rRNA sequence region and/or the ITS2rRNA sequence region.

2. The primer probe combination for mucor typing detection according to claim 1, wherein the primer pair for amplifying the rhizopus comprises the nucleotide sequence shown in SEQ ID No. 1-2, and the probe for detecting the rhizopus comprises the nucleotide sequence shown in SEQ ID No. 3;

preferably, the primer pair for amplifying the Mucor comprises nucleotide sequences shown in SEQ ID No. 4-5, and the probe for detecting the Mucor comprises nucleotide sequences shown in SEQ ID No. 6;

preferably, the primer pair for amplifying the Absidia comprises the nucleotide sequence shown in SEQ ID No. 7-8, and the probe for detecting the Absidia comprises the nucleotide sequence shown in SEQ ID No. 9;

preferably, the primer pair for amplifying the rhizomucor comprises nucleotide sequences shown in SEQ ID No. 10-11, and the probe for detecting the rhizomucor comprises the nucleotide sequence shown in SEQ ID No. 12;

preferably, the primer pair for amplifying the Cunninghamella sp comprises the nucleotide sequence shown in SEQ ID No. 13-14, and the probe for detecting the Cunninghamella sp comprises the nucleotide sequence shown in SEQ ID No. 15.

3. The primer probe combination for mucor typing detection according to claim 1 or 2, wherein the primer probe combination for mucor typing detection 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 nucleotide sequences shown in SEQ ID No. 16-17, and the probe for detecting the internal standard gene comprises nucleotide sequences shown in SEQ ID No. 18.

4. The primer-probe combination for typing detection of mucorales 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 mucor typing detection, which is characterized by comprising the primer probe combination for mucor typing detection of any one of claims 1 to 4.

6. The kit for mucor typing detection according to claim 5, wherein the kit for mucor typing detection comprises a PCR detection solution containing the primer probe combination for mucor typing detection, and the addition ratio of the upstream primer to the downstream primer in the PCR detection solution is 1 (48-52).

7. The kit for mucor typing detection according to claim 5 or 6, wherein the kit for mucor typing detection further comprises PCR mixed solution, 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 detection site sequences of rhizopus, mucor, Absidia, Rhizomucor and Cunninghamella;

preferably, the negative quality control comprises water.

8. Use of the kit for the typing detection of mucor according to any one of claims 5 to 7 for the purpose of non-disease diagnosis and/or treatment, wherein the use comprises the following steps:

extracting nucleic acid of the sample, performing fluorescence quantitative PCR amplification, analyzing a melting curve, and judging mucor in the sample and the specific typing condition thereof.

9. An apparatus for typing and detecting mucor, 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 mucor in the sample and the specific typing condition of the mucor.

10. Use of any one or a combination of at least two of the primer probe combination for mucor typing detection according to any one of claims 1 to 4, the kit for mucor typing detection according to any one of claims 5 to 7, the method for using the kit for mucor typing detection according to claim 8 for non-disease diagnosis and/or treatment purposes, or the device for mucor typing detection according to claim 9 for mucor typing detection.

Background

In recent years, with the widespread application of broad-spectrum antibiotics, corticosteroid hormones, antitumor drugs and immunosuppressants, the increase of patients with diabetes and aids, the wide use of solid organ and bone marrow transplantation, burn rescue and other life prolonging technologies provides good conditions for the propagation and pathogenesis of conditional pathogenic fungi, and the incidence of invasive mycosis infection has a remarkable increasing trend.

Mucormycosis is a fatal opportunistic fungal infection caused by mucorales (mainly rhizomucor and mucor). In fungal infections, the incidence of mucormycosis accounts for 8.3% -13%, second only to candida and aspergillus infections, and third. The fatality rate of the mucormycosis reaches 40 to 80 percent.

The diagnosis of mucormycosis infection faces a lot of difficulties, as mucormycosis is a common contaminant in laboratories or on the surface of skin mucosa, microscopic examination results obtained by directly scraping scales cannot be used as reliable evidence of tissue infiltration, and direct microscopic examination cannot be used for identification of mucormycosis species; the result identified by culture has low positive rate and long detection time; serological detection is an important detection method for clinical invasive fungal infection, G test is antigen detection aiming at 1, 3-beta-D glucan of fungal cell walls, but the cell walls of Mucor subgenus rarely express 1, 3-beta-D glucan, so the G test is not suitable for serological diagnosis of mucormycosis; galactomannan antigen (GM) test is a serological detection method for aspergillus fumigatus, which is relatively specific, but the method is relatively complicated, and the defect that mucor cannot be classified also exists.

The accurate typing detection of the mucor has important significance for treating diseases. Therefore, it is an urgent problem to provide a method for detecting mucor, which is easy to operate, short in time, and capable of being typed.

Disclosure of Invention

Aiming at the defects and the actual requirements of the prior art, the invention provides the primer probe combination for mucor typing detection 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 trichoderma typing detection, which comprises any one or a combination of at least two of a primer pair and a probe for amplifying and detecting rhizopus, a primer pair and a probe for amplifying and detecting mucor, a primer pair and a probe for amplifying and detecting absidia, a primer pair and a probe for amplifying and detecting rhizopus, and a primer pair and a probe for amplifying and detecting kummersinia;

the detection sites of the primer probe combination for mucor typing detection are positioned in the 28S rRNA sequence region and/or the ITS2rRNA sequence region.

In the invention, different primer pairs and probes are respectively designed for different mucor genera, melting curve analysis is carried out after asymmetric PCR amplification reaction, the infection condition of a sample to be detected is judged according to the change of the melting point of the probe, whether the sample is infected by mucor and the infected specific genus can be judged in the same reaction, the efficiency is high, the time consumption is short, and the detection reagent is saved; and a 28S rRNA sequence region and/or an ITS2rRNA sequence region with high conservation are/is selected as a detection site to design a primer probe, so that the specificity is high, and the result is accurate.

In the invention, the detection site sequence of rhizopus is shown as SEQ ID No.19, the detection site sequence of mucor is shown as SEQ ID No.20, the detection site sequence of Absidia is shown as SEQ ID No.21, the detection site sequence of rhizomucor is shown as SEQ ID No.22, and the detection site sequence of Cunninghamella is shown as SEQ ID No. 23.

SEQ ID No.19：

cacaaacccacacataacatttgtttatgtggtgatgggtcgcatcgctgttttattacagtgagcacctaaaatgtgtgtgattttctgtctggcttgctaggcaggaatattacgctggtctcaggatctttttttttggttcgcccaggaagtaaagtacaagagtataatccagtaactttcaaactatgatctgaagtcaggtgg。

SEQ ID No.20：

caaaccctctatccagcattttgttgaataggaatactgagagtctcctgatctattctgatctcgaacctcttgaaatgtacaaaggcctgatcttgtttaaatgcctgaacttttttttaatataaagagaagctcttgcggtaaactgtgctggggcctcccaaataatactctttttaa。

SEQ ID No.21：

tgattcccctagtaacggcgagtgaagagggaaaagctcaaagttggaacctggctgccctaggcagtccggattgtaaactaaagagcgtgattccaggcaagccggttgaccaagtcctttggaatgaggcgccactgagggtgagagccccgtaagtcgactgagcatttgtcttttgtgtttcgcgttcaaagagtcaggttgtttgggaatgcagcctaaagctggtggtaaatcccacctaaagctaaatacaggcgagagaccgatagcgaacaagtaccgtgagggaaagatgaaaagaactttgaaaagagagttaaacagtatgtgaaattgccaagagggaagcatttggagttagattgactaggagttaatcagcttggtctttggactgggtgtacttgacttcttacagtctgccaatagcagttagtcctagtggaaaaaaccagagggaaggtagtccttcgggatgtttatagacctttggaaaatacactgggattgactgaggaatgcagtagatgccactaaggcttcgtctagtgggtgctaggcaaaggtacttggtattttcagcttgctgatgtactaggttactcgagtctagtcgcctactagaactgtaatc。

SEQ ID No.22：

atcaataagcggaggaaaagaaaataacaatgatacccttagtagcggcgagcgaagtgggtaaagctcaagtttaaaacctgtttgtcctagacaaaccggattgtaaactaaggacgtgctatccaggctctttggaccttcaagtcctttggaataaggcttcacagagggtgacaatcccgtagagggtcttgaaagagtctattgcgatgcatgctccaagagtcaggttgtttgggaatgcagcctaaagtgggaggtaaatccctcctaaagctaaatattggcgagagaccgatagcaaacaagtaccgtgagggaaagttgaaaaggactttgaaaagagagtcaaaagtacgtgaaattgcttaaagggaagcgtttggagctagtttggctagtctgttatcagcctgagcttcggctttggtgtactatcaggctatttgccggccaactttcaggattggaaggaaagcttagtgctttggagtctaaagagacctttgcggaagcctctggtggagcgttggtctgccttggcccttctgaacctatagttggcttaatggctctaaacggc。

SEQ ID No.23：

atcaataagcggaggaaaagaaaataacaatgattcccctagtaacggcgagtgaagagggaaaagctcaaagttggaacctggtgggcatagctcacccggattgtaaactaaagtttttgagtcgtttagtcagccaggtaaataagtcctctggaaaggggcgacatagagggtgaaatccccgtctttggcctgagttttgattaggcgtttggcttggaaacgaagagtcaggttgtttgggaatgcagcctaaaatgggaggtaaatctctcctaaagctaaatattgacgaaagaccgatagcgaacaagtaccgtgagggaaagatgaaaagcactttgaaaagagggtcaaaaagtacgtgaaattgctgaaagggaaccgtatgaaatcagacctactggtaggtaatcaatctttcctttgggaaggatgcacttgcctgctatgtatgccagcgacattttgattgggaggaaaaaaatagtgggaatgtagcctgggcttcggcttaggtgttatagacctctataaaatactctcggttagaatgaggaacgcagcaaaccgtaaggcgaagattttaggcgcttgggggaaataattagagaatttctgcttcgggtggtgctttagttatttttttcaactcgtttgaatttcttttaatttgcttaggttgttggcttaatgattttatatgac。

Preferably, the primer pair for amplifying the rhizopus comprises nucleotide sequences shown as SEQ ID No. 1-2, and the probe for detecting the rhizopus comprises nucleotide sequences shown as SEQ ID No. 3.

Preferably, the primer pair for amplifying the Mucor comprises nucleotide sequences shown in SEQ ID No. 4-5, and the probe for detecting the Mucor comprises nucleotide sequences shown in SEQ ID No. 6.

Preferably, the primer pair for amplifying the Absidia comprises the nucleotide sequence shown in SEQ ID No. 7-8, and the probe for detecting the Absidia comprises the nucleotide sequence shown in SEQ ID No. 9.

Preferably, the primer pair for amplifying the rhizomucor comprises nucleotide sequences shown in SEQ ID No. 10-11, and the probe for detecting the rhizomucor comprises the nucleotide sequence shown in SEQ ID No. 12.

Preferably, the primer pair for amplifying the Cunninghamella sp comprises the nucleotide sequence shown in SEQ ID No. 13-14, and the probe for detecting the Cunninghamella sp comprises the nucleotide sequence shown in SEQ ID No. 15.

SEQ ID No.1：CCCACACATAACATTTGTTTAT；

SEQ ID No.2：GACAGAAAATCACACACATTTTAG；

SEQ ID No.3：AAACAGCGATGCGACCCATCACCAC；

SEQ ID No.4：GAGAGTCTCCTGATCTATTCTGA；

SEQ ID No.5：TACCGCAAGAGCTTCTCTTTA；

SEQ ID No.6：CTCGAACCTCTTGAAATGTACAAAGGCC；

SEQ ID No.7：CTAGTAACGGCGAGTGAAG；

SEQ ID No.8：GCCTCATTCCAAAGGACTTG；

SEQ ID No.9：CGGCTTGCCTGGAATCACGCTCTT；

SEQ ID No.10：TCAGGATTGGAAGGAAAGC；

SEQ ID No.11：AGAGCCATTAAGCCAACTATAG；

SEQ ID No.12：AAGGCAGACCAACGCTCCACCAGA；

SEQ ID No.13：GCTTCGGCTTAGGTGTTATAG；

SEQ ID No.14：GCCTAAAATCTTCGCCTTACG；

SEQ ID No.15：TGCTGCGTTCCTCATTCTAACCGAGAGT。

Preferably, the primer probe combination for detecting mucorales typing 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 nucleotide sequences shown in SEQ ID No. 16-17, and the probe for detecting the internal standard gene comprises nucleotide sequences shown in SEQ ID No. 18.

SEQ ID No.16：CTGAGTCGGGATGTGCTA；

SEQ ID No.17：ACCAGTATCTACCGCCAAA；

SEQ ID No.18：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 specific typing of mucor can be identified in a single channel.

In a second aspect, the present invention provides a kit for typing detection of mucor, wherein the kit for typing detection of mucor comprises the primer probe combination for typing detection of mucor described in the first aspect.

Preferably, the kit for mucor typing detection comprises a PCR detection solution containing a primer probe combination for mucor typing detection, the adding amount ratio of an upstream primer to a downstream primer in the PCR detection solution is 1 (48-52), for example, the adding amount ratio can be 1:48, 1:49, 1:50, 1:51 or 1:52, and other specific point values in the numerical value range can be selected, and are not described in detail herein.

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. 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 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. 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 mucor typing 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 detection site sequences of rhizopus, mucor, Absidia, Rhizomucor and Cunninghamella.

Preferably, the negative quality control comprises water.

In a third aspect, the present invention provides a method for using the kit for mucor typing detection in the second aspect for non-disease diagnosis and/or treatment, the method comprising the following steps:

extracting nucleic acid of the sample, performing fluorescence quantitative PCR amplification, analyzing a melting curve, and judging mucor in the sample and the specific typing condition thereof.

In a fourth aspect, the present invention provides an apparatus for typing and detecting mucor, 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 mucor in the sample and the specific typing condition of the mucor.

In a fifth aspect, the present invention provides a primer probe combination for mucor typing detection described in the first aspect, a kit for mucor typing detection described in the second aspect, a method for using the kit for mucor typing detection described in the third aspect for non-disease diagnosis and/or treatment, or a device for mucor typing detection described in the fourth aspect, or an application of at least two of the primer probe combination and the kit for mucor typing detection described in the third aspect to mucor typing detection.

Compared with the prior art, the invention has the following beneficial effects:

(1) the primer probe combination for trichoderma typing detection has good specificity and high sensitivity; the kit for mucor typing detection is convenient to use and easy to operate, and can monitor the whole detection reaction by matching with an internal standard gene primer probe, internal standard quality control, positive quality control and negative quality control, so that the accuracy and reliability of the result are ensured;

(2) the method for detecting the mucor typing does not need culture experiments, can directly use serum or alveolar lavage fluid as a sample for detection, can determine whether mucor infection occurs or not by one-time detection, can determine specific mucor by a melting curve method of asymmetric PCR, has good specificity and high sensitivity, is extremely short in time consumption, can obtain accurate results within 2h, can carry out early, rapid and accurate screening diagnosis on mucor infection and infected mucor types, has important significance on prognosis, is a new direction for mucor infection diagnosis, 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 mucor typing detection, which comprises a primer pair and a probe for amplifying and detecting rhizopus, a primer pair and a probe for amplifying and detecting mucor, a primer pair and a probe for amplifying and detecting absidia, a primer pair and a probe for amplifying and detecting rhizopus, and a primer pair and a probe for amplifying and detecting pustula;

the detection sites of the primer probe combination for mucor typing detection are positioned in the 28S rRNA sequence region and/or the ITS2rRNA sequence region.

The primer pair for amplifying the rhizopus comprises nucleotide sequences shown as SEQ ID No. 1-2, and the probe for detecting the rhizopus comprises nucleotide sequences shown as SEQ ID No. 3.

The primer pair for amplifying the Mucor comprises nucleotide sequences shown in SEQ ID No. 4-5, and the probe for detecting the Mucor comprises the nucleotide sequence shown in SEQ ID No. 6.

And the primer pair for amplifying the Absidia comprises the nucleotide sequences shown in SEQ ID No. 7-8, and the probe for detecting the Absidia comprises the nucleotide sequence shown in SEQ ID No. 9.

The primer pair for amplifying the rhizomucor comprises nucleotide sequences shown in SEQ ID No. 10-11, and the probe for detecting the rhizomucor comprises the nucleotide sequence shown in SEQ ID No. 12.

And amplifying the primer pair of the Cunninghamella to obtain a nucleotide sequence shown as SEQ ID No. 13-14, and detecting the probe of the Cunninghamella to obtain a nucleotide sequence shown as SEQ ID No. 15.

SEQ ID No.1：CCCACACATAACATTTGTTTAT；

SEQ ID No.2：GACAGAAAATCACACACATTTTAG；

SEQ ID No.3：AAACAGCGATGCGACCCATCACCAC；

SEQ ID No.4：GAGAGTCTCCTGATCTATTCTGA；

SEQ ID No.5：TACCGCAAGAGCTTCTCTTTA；

SEQ ID No.6：CTCGAACCTCTTGAAATGTACAAAGGCC；

SEQ ID No.7：CTAGTAACGGCGAGTGAAG；

SEQ ID No.8：GCCTCATTCCAAAGGACTTG；

SEQ ID No.9：CGGCTTGCCTGGAATCACGCTCTT；

SEQ ID No.10：TCAGGATTGGAAGGAAAGC；

SEQ ID No.11：AGAGCCATTAAGCCAACTATAG；

SEQ ID No.12：AAGGCAGACCAACGCTCCACCAGA；

SEQ ID No.13：GCTTCGGCTTAGGTGTTATAG；

SEQ ID No.14：GCCTAAAATCTTCGCCTTACG；

SEQ ID No.15：TGCTGCGTTCCTCATTCTAACCGAGAGT。

The primer probe combination for trichoderma typing detection further comprises a primer pair and a probe for amplifying and detecting an internal standard gene.

The primer pair for amplifying the internal standard gene comprises nucleotide sequences shown in SEQ ID No. 16-17, and the probe for detecting the internal standard gene comprises the nucleotide sequence shown in SEQ ID No. 18.

SEQ ID No.16：CTGAGTCGGGATGTGCTA；

SEQ ID No.17：ACCAGTATCTACCGCCAAA；

SEQ ID No.18：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 rhizopus is HEX, the fluorescent group of the probe for detecting mucor, Absidia, Rhizomucor and Cunninghamella is CY5, the fluorescent group of the probe for detecting internal standard gene is FAM, and the quenching groups of all the probes are BHQ 1.

In the invention, as the incidence of the rhizopus is highest, the rhizopus is singly marked, so that the detection is quicker and more accurate; the detection primers and probes of the internal standard genes are arranged, so that the detection process can be monitored, and the result is more real and credible.

Example 2

This example provides a kit for mucor typing detection, which comprises the primer probe combination for mucor typing detection in example 1.

The kit for mucor typing detection comprises PCR detection liquid containing a primer probe combination for mucor typing detection, wherein the adding amount ratio of an upstream primer to a downstream primer in the PCR detection liquid is 1: 50.

The kit for detecting mucor typing 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 plasmids containing detection site sequences of rhizopus, mucor, Absidia, Rhizomucor and Cunninghamella;

the negative quality control comprises water.

The kit for mucor typing detection is convenient to use, and the detection process can be monitored by setting internal standard quality control, positive quality control and negative quality control, so that the accuracy of detection reaction is ensured.

Example 3

In this example, the kit for typing and detecting mucor prepared in example 2 is used to detect a sample, and the specific steps are as follows:

(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 53 ℃ 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 mucor in the sample and the specific typing condition of the mucor.

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 the fluorescent probe in combination with the melting curve in example 3 can clearly distinguish the infection of four Mucor fungi in the same channel, wherein the Tm value of Mucor is 60.5-61.5; the Tm value of the Absidia is 63-64.5; the Tm value of Rhizomucor is 66.5-67.5; the Tm value of the Cunninghamella is 69.5-70.5, and all fluorescence values are very similar. Example 4 cannot distinguish between Absidia and Mucor, Rhizomucor and Cunninghamella, and the Tm values are very close and are not easily distinguished. Example 5 still did not distinguish between Absidia and Mucor, Rhizomucor and Cunninghamella, which had very similar Tm values and were not easily distinguishable. In example 6, the fluorescence values of the detected Absidia and Cunninghamella sp.were low, and Absidia and Mucor, Rhizomucor and Cunninghamella sp.could not be distinguished, and their Tm values were very 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 invention provides a primer probe combination for typing detection of mucor, and the primer probe combination has good specificity and sensitivity; the kit for performing mucor typing detection, which contains the primer probe combination for performing mucor typing detection, is convenient to use, can confirm whether a sample is infected by mucor and the infected specific mucor in one detection reaction by matching with a corresponding use method, can simultaneously detect and analyze multiple mucors in the same fluorescence channel through a melting curve, is short in time consumption and high in detection efficiency, guarantees the accuracy of results by setting multiple quality control products, can save reagents, and has a wide application prospect.

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 type detection of mucor and application thereof

<130> 2021

<160> 23

<170> PatentIn version 3.3

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<213> Artificial sequence

<400> 8

gcctcattcc aaaggacttg 20

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<400> 9

cggcttgcct ggaatcacgc tctt 24

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence

<400> 10

tcaggattgg aaggaaagc 19

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<400> 11

agagccatta agccaactat ag 22

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<400> 12

aaggcagacc aacgctccac caga 24

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence

<400> 13

gcttcggctt aggtgttata g 21

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<400> 14

gcctaaaatc ttcgccttac g 21

<210> 15

<211> 28

<212> DNA

<213> Artificial sequence

<400> 15

tgctgcgttc ctcattctaa ccgagagt 28

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence

<400> 16

ctgagtcggg atgtgcta 18

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence

<400> 17

accagtatct accgccaaa 19

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence

<400> 18

acggaaccat tcgctcgcca atcg 24

<210> 19

<211> 210

<212> DNA

<213> Rhizopus

<400> 19

cacaaaccca cacataacat ttgtttatgt ggtgatgggt cgcatcgctg ttttattaca 60

gtgagcacct aaaatgtgtg tgattttctg tctggcttgc taggcaggaa tattacgctg 120

gtctcaggat cttttttttt ggttcgccca ggaagtaaag tacaagagta taatccagta 180

actttcaaac tatgatctga agtcaggtgg 210

<210> 20

<211> 183

<212> DNA

<213> Mucor

<400> 20

caaaccctct atccagcatt ttgttgaata ggaatactga gagtctcctg atctattctg 60

atctcgaacc tcttgaaatg tacaaaggcc tgatcttgtt taaatgcctg aacttttttt 120

taatataaag agaagctctt gcggtaaact gtgctggggc ctcccaaata atactctttt 180

taa 183

<210> 21

<211> 640

<212> DNA

<213> Absidia

<400> 21

tgattcccct agtaacggcg agtgaagagg gaaaagctca aagttggaac ctggctgccc 60

taggcagtcc ggattgtaaa ctaaagagcg tgattccagg caagccggtt gaccaagtcc 120

tttggaatga ggcgccactg agggtgagag ccccgtaagt cgactgagca tttgtctttt 180

gtgtttcgcg ttcaaagagt caggttgttt gggaatgcag cctaaagctg gtggtaaatc 240

ccacctaaag ctaaatacag gcgagagacc gatagcgaac aagtaccgtg agggaaagat 300

gaaaagaact ttgaaaagag agttaaacag tatgtgaaat tgccaagagg gaagcatttg 360

gagttagatt gactaggagt taatcagctt ggtctttgga ctgggtgtac ttgacttctt 420

acagtctgcc aatagcagtt agtcctagtg gaaaaaacca gagggaaggt agtccttcgg 480

gatgtttata gacctttgga aaatacactg ggattgactg aggaatgcag tagatgccac 540

taaggcttcg tctagtgggt gctaggcaaa ggtacttggt attttcagct tgctgatgta 600

ctaggttact cgagtctagt cgcctactag aactgtaatc 640

<210> 22

<211> 586

<212> DNA

<213> Rhizomucor

<400> 22

atcaataagc ggaggaaaag aaaataacaa tgataccctt agtagcggcg agcgaagtgg 60

gtaaagctca agtttaaaac ctgtttgtcc tagacaaacc ggattgtaaa ctaaggacgt 120

gctatccagg ctctttggac cttcaagtcc tttggaataa ggcttcacag agggtgacaa 180

tcccgtagag ggtcttgaaa gagtctattg cgatgcatgc tccaagagtc aggttgtttg 240

ggaatgcagc ctaaagtggg aggtaaatcc ctcctaaagc taaatattgg cgagagaccg 300

atagcaaaca agtaccgtga gggaaagttg aaaaggactt tgaaaagaga gtcaaaagta 360

cgtgaaattg cttaaaggga agcgtttgga gctagtttgg ctagtctgtt atcagcctga 420

gcttcggctt tggtgtacta tcaggctatt tgccggccaa ctttcaggat tggaaggaaa 480

gcttagtgct ttggagtcta aagagacctt tgcggaagcc tctggtggag cgttggtctg 540

ccttggccct tctgaaccta tagttggctt aatggctcta aacggc 586

<210> 23

<211> 711

<212> DNA

<213> Klebsiella sp

<400> 23

atcaataagc ggaggaaaag aaaataacaa tgattcccct agtaacggcg agtgaagagg 60

gaaaagctca aagttggaac ctggtgggca tagctcaccc ggattgtaaa ctaaagtttt 120

tgagtcgttt agtcagccag gtaaataagt cctctggaaa ggggcgacat agagggtgaa 180

atccccgtct ttggcctgag ttttgattag gcgtttggct tggaaacgaa gagtcaggtt 240

gtttgggaat gcagcctaaa atgggaggta aatctctcct aaagctaaat attgacgaaa 300

gaccgatagc gaacaagtac cgtgagggaa agatgaaaag cactttgaaa agagggtcaa 360

aaagtacgtg aaattgctga aagggaaccg tatgaaatca gacctactgg taggtaatca 420

atctttcctt tgggaaggat gcacttgcct gctatgtatg ccagcgacat tttgattggg 480

aggaaaaaaa tagtgggaat gtagcctggg cttcggctta ggtgttatag acctctataa 540

aatactctcg gttagaatga ggaacgcagc aaaccgtaag gcgaagattt taggcgcttg 600

ggggaaataa ttagagaatt tctgcttcgg gtggtgcttt agttattttt ttcaactcgt 660

ttgaatttct tttaatttgc ttaggttgtt ggcttaatga ttttatatga c 711