CN115820921A

CN115820921A - Primer probe combination for detecting three kinds of lung invasive fungi and digital PCR kit thereof

Info

Publication number: CN115820921A
Application number: CN202211632077.1A
Authority: CN
Inventors: 夏江; 高学娟
Original assignee: Pilot Gene Technologies Hangzhou Co ltd
Current assignee: Pilot Gene Technologies Hangzhou Co ltd
Priority date: 2022-12-19
Filing date: 2022-12-19
Publication date: 2023-03-21
Anticipated expiration: 2042-12-19
Also published as: CN115820921B

Abstract

The invention relates to the technical field of biology, in particular to a primer probe combination for detecting three lung invasive fungi and a digital PCR detection kit thereof. The primer probe combination designed based on the specific conserved region of the mitochondrial gene of the pathogenic bacteria can realize the rapid detection of three pathogenic bacteria of aspergillus fumigatus, cryptococcus neoformans and yersinia sporogenes, and has good accuracy and specificity and high sensitivity.

Description

Primer probe combination for detecting three kinds of lung invasive fungi and digital PCR kit thereof

Technical Field

The invention relates to the technical field of biology, in particular to a primer probe combination for detecting three lung invasive fungi and a digital PCR kit thereof.

Background

Aspergillus is the most infectious mould species, and 3176 of the mould species and 1378 of the mould species have been found, wherein more than 250 of the broad-spectrum nature 40 are pathogenic bacteria. Aspergillus can parasitize the skin and upper respiratory tract of normal people, and is a conditional pathogen. The normal people have certain resistance to aspergillus and do not cause diseases. Aspergillosis is mostly secondary, when the resistance of an organism is reduced, pathogenic bacteria can pass through a skin mucous membrane injury part or be inhaled into a respiratory tract, and then enter blood circulation to other tissues or organs to cause diseases. Patients with immunosuppression, malignancy, organ transplantation (common in lung transplantation) are the major patient population with aspergillus infection. The main infection sites of aspergillus are the lungs, which are divided into three main groups: allergic bronchial infection, chronic infection, and invasive infection. The aspergillus mainly infecting the lung includes aspergillus fumigatus, aspergillus flavus, aspergillus niger and the like. The tobacco aspergillosis accounts for more than 90% of all aspergillosis.

Cryptococcosis is a pulmonary or disseminated infectious disease caused by cryptococci, mainly resulting in pneumonia and meningitis, and also in infections of the skin, bones or internal organs. There are more than 70 cryptococci found so far, of which 7 infect humans: two subspecies of cryptococcus neoformans and 5 subspecies of cryptococcus gatherens, with cryptococcosis neoformans accounting for about 90% of all cryptococcosis. The cryptococcosis population is mainly immunodeficiency patients, such as HIV infection, malignant tumor, stem cell or organ transplantation, autoimmune disease and nervous system complications.

Yersinia pneumonitis pneumonia is pneumonia caused by pneumocystis, and there are two main groups of infections: HIV virus infected and non-HIV virus infected, HIV virus infected persons are progressing slowly; non-HIV virus infected persons are mainly: the patient with malignant blood disease, organ transplantation receptor and cancer chemotherapy patient are characterized in that: rapid progress, high incidence of respiratory failure and high mortality.

Mitochondria, also known as the energy supply station of eukaryotic cells, are an important organelle in eukaryotic cells because aerobic respiration occurs within mitochondria. As a semi-autonomous organelle, mitochondria possess a certain number of genetic systems, called mitochondrial genes, independent of the nucleus.

Because fungi are difficult to culture and grow slowly, the main method for detecting aspergillus infection at present is GM detection, and the defect of the GM detection is that the GM detection can cause false positive results under the influence of certain foods or medicines; the cryptococcus is clinically diagnosed by combining clinical manifestations and microscopic examination results, and is confirmed by fungus culture or tissue staining; the pneumocystis yezoensis is clinically diagnosed by combining clinical manifestations and microscopic examination results. There are also several patents for detecting aspergillus, cryptococcus and yersinia by utilizing qPCR technology, but the detection targets of the patents are genomic DNAs (18S RNA, ITS region or 28S RNA) of one species of aspergillus fumigatus or three species of aspergillus fumigatus, cryptococcus or yersinia pneumocystis, and no report for joint detection of the above several species by utilizing a linear gene as the detection target exists at present.

Disclosure of Invention

In view of this, the invention provides a primer probe combination for detecting three kinds of lung invasive fungi and a digital PCR detection kit thereof. The primer probe combination is combined with a digital PCR technology, can quickly realize the combined detection of aspergillus fumigatus, yersinia sporogenes and cryptococcus neoformans, and has good accuracy, high sensitivity and short detection time.

In order to achieve the above object, the present invention provides the following technical solutions:

a primer probe combination for detecting three lung invasive fungi comprising:

primers and probes for detecting aspergillus: 1-2 and 3, respectively;

and (3) primers and probes for detecting cryptococcus neoformans: primers of the nucleotide sequences shown in SEQ ID NO. 4-5 and probes of the nucleotide sequences shown in SEQ ID NO. 6; and/or

Primers and probes for detecting yersinia sporogenes: primers of the nucleotide sequences shown in SEQ ID NO. 7-8 and probes of the nucleotide sequences shown in SEQ ID NO. 9.

The primer probe combination is designed aiming at specific conserved regions of mitochondrial genes of aspergillus fumigatus, cryptococcus neoformans and yersinia sporogenes respectively, wherein nucleotide sequences of the conserved regions of the mitochondrial genes of aspergillus fumigatus, cryptococcus neoformans and yersinia sporogenes are shown as SEQ ID NO: 14-16 in sequence.

In the invention, the 5 'end of the probe is marked with a fluorescent group, and the 3' end of the probe is marked with a quenching group;

the fluorescent group is selected from FAM, ROX, VIC, CY5, ATTO-425, HEX, TAMRA, CY5.5, CY7, ATTO-488, ATTO-550, ATTO-565, ATTO-590, ATTO-635, ATTO-532, ATTO-549 and ATTO-633; the quenching group is selected from MGB or BHQ1, BHQ2 and BHQ3.

In some embodiments:

the 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 3 is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group MGB;

the 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 6 is marked with a fluorescence reporter group ROX, and the 3' end is marked with a fluorescence quenching group MGB;

the 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 9 is marked with a fluorescence reporter group CY5, and the 3' end is marked with a fluorescence quenching group MGB.

The invention also provides application of the primer probe combination in preparing a lung disease diagnosis kit. The pulmonary disease is a disease induced by infection with at least one pathogenic bacterium selected from the group consisting of Aspergillus fumigatus, cryptococcus neoformans and Chrysosporium yerianum.

The invention also provides a kit for diagnosing lung diseases, which comprises the primer probe combination.

In some embodiments, the kit of the present invention further comprises primers and probes designed for the conserved region of the internal reference IC (the sequence shown in SEQ ID No. 13): the primer sequence is shown as SEQ ID NO. 10-11; the probe sequence is shown in SEQ ID NO. 12. In some embodiments, the probe shown in SEQ ID NO. 12 is labeled with a fluorescent reporter group VIC at the 5 'end and a fluorescent quencher group BHQ1 at the 3' end.

The invention also provides a method for detecting lung pathogenic bacteria, which utilizes the primer probe combination or the kit to carry out digital PCR detection on a sample to be detected.

The program for digital PCR detection comprises: pre-denaturation at 98 ℃ for 5min; denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 30 seconds, and circulation for 40 times.

In the invention, the samples to be detected comprise human sputum samples, alveolar lavage fluid samples, blood samples and thoracoabdominal water samples. In some embodiments, the assays of the invention are assays for non-diagnostic purposes, such as assays on samples of an environment or on food samples, wherein the samples in the environment comprise water samples or swabs on the surface of an object.

The primer and the probe are designed aiming at the specific region of the mitochondrial genes of aspergillus fumigatus, yersinia sporogenes and cryptococcus neoformans, can quickly realize the joint detection of three pulmonary pathogenic bacteria of aspergillus fumigatus, yersinia sporogenes and cryptococcus neoformans, and has the characteristics of high sensitivity, good specificity and high repeatability.

Detailed Description

The invention provides a primer probe combination for detecting three kinds of lung invasive fungi and a digital PCR detection kit thereof. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 System and method for primer probe screening and digital PCR detection of the invention

(first) screening of primer probes

6 pairs of primer probes are designed for each of the three bacteria, qPCR is carried out by taking plasmids of corresponding sequences of 10000 copies/mu l (stock solution), 1000 copies/mu l (10-fold dilution) and 100 copies/mu l (100-fold dilution) as templates, and the CT value and the maximum fluorescence value of 6 pairs of Aspergillus fumigatus detection are as follows:

TABLE 1

Because of the maximum fluorescence intensity of the amplification product, the difference between the gray values of the negative and positive droplets is determined in the droplet test digital PCR, in order to ensure the maximization of the difference between the gray values of the negative and positive droplets, a primer probe combination with the maximum fluorescence intensity of the amplification product larger is selected during the primer probe selection, and the third pair and the fourth pair can be selected by screening the primer probe combinations of 6 pairs of aspergillus fumigatus under the condition. Because 10 is about 3.3 times of 2, the detected CT value is increased by 3.3 for every 10 times of reduction of the template concentration, and the combination is considered to be the combination with the minimum influence by external conditions and can be selected for building a multiplex system. From these two factor analyses, primer probe combinations for 6 pairs of A.fumigatus we chose the third pair for the set-up of the system. Similarly, we selected the second and fifth pairs from 6 pairs of Cryptococcus neoformans and Yersinia sporogenes primer probes, and the specific sequences of the screened primer probes and their targets are shown in Table 2, and the sequences of the control primer probes of one pair are shown in Table 3.

TABLE 2

TABLE 3 control primer Probe sequences

(II) digital PCR detection system and method

1. Sample(s)

1.1 bacterial DNA sample: bacterial DNA samples: and (3) externally purchasing dead bacteria, and extracting DNA by a magnetic bead method.

1.2 human DNA samples: extracting genome DNA by a magnetic bead method by using sterile sputum/alveolar lavage fluid. 1.3 exogenous internal reference template: synthesized by biological companies, and dissolved and diluted for later use.

2 digital PCR amplification system

2.1 25 xBc primer probe mixture

TABLE 4

TABLE 5ddPCR reaction System

Reagent	Volume (μ L)	Final concentration
			Water (W)	4.85
5×ddPCR Mix	3	1×
			IC-S	1
DNA	5
			25 xBc primer probe mixture	1.15
Total up to	15

2.2 amplification procedure

98℃5min【98℃15s，60℃30s】*40。

Example 2 specific detection

The following samples were tested with the primer probes of table 2 of example 1, and the results were as follows:

TABLE 6

The result shows that the primer probe combination has good specificity for detecting various common bacterial fungi, and has good stability for detecting three repetitions of aspergillus fumigatus, cryptococcus neoformans and cryptosporidium jejuni.

Example 3 detection of amplification efficiency

The amplification efficiency of the primer probes of tables 2 and 3 was examined, and the results are shown in Table 7.

TABLE 7

The results show that the Ct value increases by about 3 for each 10-fold decrease in primer probe concentration as a result of qPCR with the optimal primer probe concentrations screened in Table 4 and the concentrations of the above 3 artificially synthesized DNA templates, respectively. Since the power of 3.3 of 2 is equal to 10, the amplification efficiency of all primer probes in the system is close to 100%.

Example 4

Sputum samples positive for 4 cases of aspergillus fumigatus, 4 cases of cryptococcus neoformans and 4 cases of yersinia pneumocystis were tested using the primer probe system of the present invention and the 28sRNA primer probes (28 sRNA and a. Fum-MIT) shown in table 3. And extracting DNA of the sputum sample, and respectively adding 5 mu l of DNA into each system as a template.

The measurement values of Aspergillus fumigatus are shown in Table 7.

TABLE 7

	Sample 1 (FAM)	Sample 2 (FAM)	Sample 3 (FAM)	Sample 4 (FAM)
					28sRNA	3.11copies/μl	2.57copies/μl	2.62copies/μl	3.78copies/μl
A.FUM-MIT	1287copies/μl	1001copies/μl	1064copies/μl	1479copies/μl

In real sample detection, the detection concentration taking aspergillus fumigatus mitochondrial gene as a detection target is about 390 times that taking 28sRNA as the detection target, the detection rate of a positive sample is higher, and the detection concentration taking 28sRNA as the detection target is lower, so that a false negative result is easy to appear.

The values for Cryptococcus neoformans are shown in Table 8.

TABLE 8

	Sample 1 (FAM)	Sample 2 (FAM)	Sample 3 (FAM)	Sample 4 (FAM)
					28sRNA	24.31copies/μl	8.62copies/μl	7.49copies/μl	13.65copies/μl
C.NEO-MIT	233.62copies/μl	87.52copies/μl	76.23copies/μl	138.23copies/μl

In the real sample detection, the detection concentration taking the cryptococcus neoformans mitochondrial gene as the detection target is about 10 times that taking 28sRNA as the detection target, the detection rate of a positive sample is higher, and the detection concentration taking 28sRNA as the detection target is lower, so that a false negative result is easy to appear.

The values for Yersinia pneumonocystis are shown in Table 9.

TABLE 9

	Sample 1 (FAM)	Sample 2 (FAM)	Sample 3 (FAM)	Sample 4 (FAM)
					28sRNA	24.31copies/μl	8.62copies/μl	7.49copies/μl	13.65copies/μl
IF-PNEU	972.36copies/μl	578.12copies/μl	301.49copies/μl	587.26copies/μl

In real sample detection, the detection concentration of the yersinia sporogenes serving as a detection target is about 40 times that of 28sRNA serving as the detection target, the detection rate of a positive sample is higher, and the detection concentration of 28sRNA serving as the detection target is lower, so that a false negative result is easy to appear.

Example 5 blank detection Limit (LoB) values

20 parts of deionized water was extracted daily as a template, and the primer probe system (28 sRNA and mitochondrial gene) of the present invention was used on different amplification instruments for three consecutive days, once a day. Except that all detection values are 0, analyzing whether the detection result is in accordance with normal distribution by using a ss23.0 software S-W, and if the detection result is in accordance with the normal distribution, analyzing the LoB value by using a parameter method; if the distribution is not normal, analyzing the LoB value by a nonparametric method. The results were as follows:

watch 10

The maximum value of the results of 3 times of calculation using mitochondrial genes as detection targets is the LoB value, namely 0.09 copies/mu l of aspergillus fumigatus, 0.11 copies/mu l of cryptococcus neoformans and 0 copies/mu l of non-sporophyte yeri; the maximum of 3 calculations with 28sRNA as the detection target was the LoB value, i.e., 0.29copies/μ l for Aspergillus fumigatus, 0.19copies/μ l for Cryptococcus neoformans, and 0.21copies/μ l for yarrowia. The result shows that the specificity of the primer probe system taking the mitochondrial gene as the detection target is obviously higher than that of the primer probe system taking 28sRNA as the detection target.

Example 6 lowest detection Limit (LoD) value

1.1 minimum detection limit (LoD)

If LoB =0, adopting a probability unit scheme; if LoB ≠ 0, the classical scheme is employed. The largest LOD of 3 reagent batches was formulated as the final reported value.

1.1.1 probability Unit scheme

Using ultrapure water as a substrate, DNAs of different target bacteria at known concentrations were incorporated, respectively, to prepare positive specimens. Then, the samples were diluted with ultrapure water in a gradient manner.

The same set of instruments including a droplet generator, a PCR amplification instrument and a chip reader are used, and 3 batch reagents are used for respectively and repeatedly detecting different concentration gradients of each target bacterium sample for 30 times.

And respectively calculating the positive detection rate of each concentration gradient of each target bacteria specimen, and selecting the lowest concentration with the positive detection rate of more than or equal to 95 percent as the LoD value of each target bacteria.

1.1.2 classical protocol

Using ultrapure water as a substrate, DNAs of different target bacteria at known concentrations were incorporated, respectively, to prepare positive specimens. Then, the samples were diluted with respective ultrapure water to five concentrations of 1 × LoB, 2 × LoB, 3 × LoB, 4 × LoB, and 5 × LoB, and used as low-value specimens.

The same set of instruments including a droplet generation instrument, a PCR amplification instrument and a chip reader are used, 3 batch number reagents are used, five low-value samples of each target bacterium are respectively and repeatedly detected for at least 12 times, and the total number of the measurement results of at least 60 low-value samples is measured.

Respectively calculating LoD values of the target bacteria, wherein the method comprises the following steps:

1) The data distribution of at least 60 low value specimen measurements per lot of reagent is analyzed.

2) If the data are normally distributed, a parameter statistical method is adopted for analysis.

3) If the data are distributed in a biased way, a nonparametric statistical method is adopted for analysis.

The limit of detection is the LoD value, and the results are shown in the following table:

TABLE 11

The results show that the sensitivity of the primer probe system (table 2) using mitochondrial genes as detection targets is obviously higher than that of the primer probe system using 28sRNA as detection targets.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A primer probe combination for detecting three lung invasive fungi, comprising:

primers and probes for detecting aspergillus: 1-2 and 3 respectively;

Primers and probes for detecting yersinia sporogenes: primers of nucleotide sequences shown in SEQ ID NO. 7-8 and probes of nucleotide sequences shown in SEQ ID NO. 9.

2. The primer probe combination of claim 1, wherein the probe is labeled with a fluorescent group at the 5 'end and a quenching group at the 3' end;

3. The primer-probe combination according to claim 2,

4. Use of the primer probe combination of any one of claims 1 to 3 for the preparation of a kit for the diagnosis of pulmonary diseases.

5. The use of claim 4, wherein the pulmonary disease is a disease induced by infection with at least one pathogenic bacterium selected from the group consisting of Aspergillus fumigatus, cryptococcus neoformans and Pneumocystis yeri.

6. A kit for diagnosing a pulmonary disease, comprising the primer probe combination according to any one of claims 1 to 3.

7. The kit of claim 6, further comprising primers and probes for detecting an internal reference gene: the primer sequence is shown as SEQ ID NO 10-11; the probe sequence is shown in SEQ ID NO. 12.

8. The kit according to claim 7, wherein the probe represented by SEQ ID NO. 12 is labeled at the 5 'end with a fluorescence reporter group VIC and at the 3' end with a fluorescence quencher group BHQ1.

9. A method for the detection of pathogenic bacteria in the lung for non-diagnostic purposes, characterized in that a sample to be tested is subjected to digital PCR detection using the primer probe set according to any one of claims 1 to 3 or the kit according to any one of claims 6 to 8.

10. The method of claim 9, wherein the digital PCR detection procedure comprises: pre-denaturation at 98 ℃ for 5min; denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 30 seconds, and circulation for 40 times.