CN117512204A

CN117512204A - Primer and probe combination for multiplex detection of aspergillus, cryptococcus and yersinia, kit and application

Info

Publication number: CN117512204A
Application number: CN202410016952.6A
Authority: CN
Inventors: 陈海琴; 李婷婷; 陈冬冬; 李翔
Original assignee: Jiangsu Mics Medical Technology Co ltd
Current assignee: Jiangsu Mics Medical Technology Co ltd
Priority date: 2024-01-05
Filing date: 2024-01-05
Publication date: 2024-02-06
Anticipated expiration: 2044-01-05
Also published as: CN117512204B

Abstract

The invention provides a combination of a primer and a probe for multiplex detection of aspergillus, novel cryptococcus and yersinia pneumospores, a kit and application, wherein the aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), the novel cryptococcus and the yersinia pneumospores are taken as detection targets, the primer and the probe are designed, and the multiplex quantitative PCR fungus detection kit is prepared, so that the kit is suitable for various detection samples, has high sensitivity and good specificity, has strong fluorescent signals, and provides reliable diagnosis guarantee for clinical detection of fungus infection.

Description

Primer and probe combination for multiplex detection of aspergillus, cryptococcus and yersinia, kit and application

Technical Field

The invention relates to the technical field of fungus detection, in particular to a primer probe combination and a detection kit of aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumoconima and application thereof.

Background

Invasive mycoses (invasive fungal disease, IFD), also known as invasive fungal infections, refer to pathological changes and pathophysiological processes in which fungi invade human tissue, blood, and grow and reproduce therein leading to tissue damage, organ dysfunction and inflammatory responses. Aspergillus, cryptococcus, yersinia, are causative bacteria of invasive mycosis.

Aspergillosis is a mycosis caused by infection of aspergillosis, and can involve multiple organ systems such as skin, mucous membrane, eyes, nose, bronchi, lung, gastrointestinal tract, nervous system, bones and the like, and severe cases of aspergillosis are distributed worldwide. The pathogen Aspergillus spp is a common conditionally pathogenic fungus, widely distributed in nature, and not uncommon for its infectors. Aspergillus predisposes to infection of open cavities such as lung cavities, sinuses or external auditory meatuses (otomycoses) caused by pulmonary diseases such as bronchiectasis, tumors and tuberculosis. Infection at these sites mainly causes locally invasive destructive lesions, although systemic infections may also be caused, especially in immunocompromised patients with neutropenia, with corticosteroids. Pathogens commonly found in clinical aspergillosis include aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, and the like.

The most commonly caused infections of cryptococcus (Cryptococcus neoformans) are cryptococcoid meningitis and cryptococcosis pneumonia, and in addition, diseases such as blood flow infection, endocarditis, myocarditis, cellulitis, peritonitis, intestinal perforation, endophthalmitis, arthritis, osteomyelitis, necrotizing fasciitis, infectious shock, pyogenic infection, abscess and the like can be caused, and cryptococcosis is well developed in people with low immunity. In cryptococcus infection, the infection of central nervous system patients with basic diseases and hypoimmunity is mainly caused, and in pulmonary infection patients, the infection incidence frequency of normal immunity patients is increased year by year, which suggests that hypoimmunity is no longer a necessary condition in cryptococcus infection patients.

Yersinia pneumospores (Pneumocystis jiroveci) are a conditional pathogen of the respiratory system, the current mode of transmission is not clear, but studies have demonstrated that it is possible to transmit by aerosol. Long-term incubation of yersinia pneumospores in the trachea, bronchi or alveolar spaces can form a recessive infection, asymptomatic. Latent or newly infected yersinia pneumospores proliferate in large numbers when the host is hypoimmunity, invade the lungs, form yersinia pneumonitis, and appear as diffuse interstitial changes in the lungs. In people with low body immunity caused by combined AIDS infection, malignant tumor, organ transplantation postoperative, long-term large-dose application of immunosuppressant, malignant malnutrition, etc., yersinia pneumoconiosis is multiple.

According to the "laboratory diagnostic methods for invasive mycoses clinical application expert consensus" (2022 edition), the current clinical tests for invasive mycoses can be divided into two categories, histological and microbiological tests. Although the histological examination can directly obtain pathogenic bacteria at an infection site, the culture step is still needed when the pathogenic bacteria load is low, the time consumption is long, and the method is not suitable for early diagnosis. In microbiological examination, direct microscopic examination has the problems of false negative, low sensitivity and the like. Fungus culture identification also has the problem of low positive rate and takes longer time. The G test and GM test in serological tests are widely used for early screening and continuous detection of aspergillus infections, but in the presence of false positives.

Currently, there are few applications or related reports of joint inspection qPCR reagents of Aspergillus (including Aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, etc.), novel Cryptococcus, and Yersinia pneumospores based on molecular biological methods. Therefore, a reagent for detecting the pathogen combination in the field needs a rapid and accurate molecular biological method, so that early screening and early diagnosis of invasive mycosis are realized, and intervention treatment and disease control are facilitated in time.

Disclosure of Invention

The invention aims to solve the technical problem of providing a kit for detecting aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumoconiosis by multiplex quantitative PCR so as to solve the problem of insufficient kits for multiplex detection of the target combination in the prior art, wherein the combination specificity of the used primer and probe is higher and the sensitivity is higher.

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

the invention provides a combination of a primer and a probe for detecting aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia, wherein the aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia detection primer probe set takes the 104 th-131 th site on a conserved DNA fragment in the 18s gene of the aspergillus as a detection target, the 104 th-131 th site on a conserved DNA fragment in the cap10 gene of the novel cryptococcus as a detection target and the 104 th-131 th site on a conserved DNA fragment in the Dhfr gene of the yersinia as a detection target. And respectively designing primers according to the target region sequences, and screening to finally obtain the primers.

The invention provides a combination of a primer and a probe for detecting aspergillus, wherein the nucleotide sequence of the primer is shown as SEQ ID No. 1-2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.

In some embodiments, the aspergillus includes any species of aspergillus common in the art. Preferably, the method comprises Aspergillus fumigatus, aspergillus flavus, aspergillus terreus and Aspergillus niger.

The invention provides a combination of a primer and a probe for detecting novel cryptococcus, wherein the nucleotide sequence of the primer is shown as SEQ ID No. 4-5, and the nucleotide sequence of the probe is shown as SEQ ID No. 6.

The invention provides a combination of a primer and a probe for detecting yersinia pneumoconiosis, wherein the nucleotide sequence of the primer is shown as SEQ ID No. 7-8, and the nucleotide sequence of the probe is shown as SEQ ID No. 9.

In some embodiments, the combination of aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, etc.), cryptococcus neoformans, yersinia pneumospore detection primers and probes comprises:

based on the nucleotide sequence shown in any of SEQ ID NO. 1-9, deleting or adding one or more nucleotides to obtain the derivative primer or probe of the nucleotide sequence with the same function as the nucleotide sequence.

based on the nucleotide sequence shown in any of SEQ ID NO. 1-9, the derivative primer or probe of the nucleotide sequence with the same function as the nucleotide sequence is obtained through nucleotide substitution or modification.

derivative primers or probes which have more than 80% homology with the nucleotide sequences shown in any of SEQ ID NO. 1-9 or are functionally similar to the nucleotide sequences shown in any of SEQ ID NO. 1-9.

a primer or probe complementary to any of the nucleotide sequences shown in SEQ ID NO. 1-9.

The invention provides a primer and probe combination for multiplex quantitative PCR detection of aspergillus, novel cryptococcus and yersinia pneumospore, which comprises the following steps:

primer 18S-F1 of the nucleotide sequence shown in SEQ ID No.1, primer 18S-R5 of the nucleotide sequence shown in SEQ ID No. 2, probe 18S-P1-ROX of the nucleotide sequence shown in SEQ ID No. 3;

primer cap10-F3 of nucleotide sequence shown in SEQ ID No. 4, primer cap10-R2 of nucleotide sequence shown in SEQ ID No. 5, probe cap10-P1-FAM of nucleotide sequence shown in SEQ ID No. 6;

primer dhfr-F3 of the nucleotide sequence shown in SEQ ID No. 7, primer dhfr-R5 of the nucleotide sequence shown in SEQ ID No. 8, probe dhfr-P1-CY5 of the nucleotide sequence shown in SEQ ID No. 9.

In some embodiments, any one of the combinations of primers and probes provided herein, the 5 'end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group; wherein the fluorescent reporter group is selected from any one of FAM, VIC, HEX, CY, CY3, JOE and ROX; the fluorescence quenching group is selected from any one of BHQ1, BHQ2, BHQ3 and TAMRA.

In some embodiments, the nucleotide sequence depicted in SEQ ID No.3 is 5 'tagged with a fluorescent group ROX and 3' tagged with a quenching group BHQ2.

In some embodiments, the 5' end of the probe of the nucleotide sequence set forth in SEQ ID No.6 is labeled with a fluorophore FAM; the 3' -end marks the quenching group BHQ2.

In some embodiments, the probe of the nucleotide sequence shown in SEQ ID No.9 is labeled with a fluorescent group CY5 at the 5 'end and a quenching group BHQ3 at the 3' end.

In some embodiments, in any one of the primer and probe combinations provided by the present invention, the nucleotide sequence shown in SEQ ID No.3 has a 5 'end labeled with a fluorescent group ROX and a 3' end labeled with a quenching group BHQ2; marking a fluorescent group FAM at the 5' -end of the probe with the nucleotide sequence shown in SEQ ID No. 6; 3' end marks quenching group BHQ2; the 5 '-end of the probe of the nucleotide sequence shown in SEQ ID No.9 is marked with a fluorescent group CY5, and the 3' -end is marked with a quenching group BHQ3.

In another aspect, the invention provides a kit for detecting at least one pathogenic microorganism of aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, etc.), cryptococcus neoformans, and yersinia pneumoconica: combinations comprising the above primers and probes.

In another aspect, the invention provides a kit for multiplex quantitative PCR detection of Aspergillus, cryptococcus neoformans, yersinia pneumospores, comprising a combination of any of the above primers and probes.

Further, in some embodiments, the kits provided herein include an amplification reaction liquid mixture (also referred to as "amplification reaction liquid MIX", "PCR reaction MIX"), a positive quality control, and a negative quality control.

Further, the amplification reaction mixture MIX includes a PCR buffer, dNTP solution, taq dna polymerase, and UDG enzyme.

Further, the PCR buffer comprises Tris-HCl and MgCl ₂ KCl and Triton X-100, tris-HCl concentration of 10-50 mM in PCR buffer ₂ The concentration of (2) is 3-6 mM, the concentration of KCl is 10-50 mM, and the concentration of Triton X-100 is 1% -10% (v/v).

Further, the dNTP solution comprises dATP, dTTP, dCTP, dGTP and dUTP, wherein dATP concentration in the dNTP solution is 0.1-0.3 mM, dTTP concentration in the dNTP solution is 0.1-0.3 mM, dCTP concentration in the dCTP solution is 0.1-0.3 mM, dGTP concentration in the dGTP solution is 0.1-0.3 mM, and dUTP concentration in the dCTP solution is 0.1-0.3 mM.

Further, the single detection reaction liquid contains 1.5U-3U of TaqDNA polymerase and 0.5U-2U of UDG enzyme.

In some embodiments, the final concentration of the primer 18S-F1, the primer 18S-R5, the primer cap10-F3, the primer cap10-R2, the primer dhfr-F3 and the primer dhfr-R5 is independently 0.1-0.5 mu M respectively; the final concentrations of the probe 18S-P1-ROX, the probe cap10-P1-FAM and the probe dhfr-P1-CY5 are independently 0.05-0.25 mu M respectively.

In some embodiments, the invention provides any of the kits further comprising a positive quality control and a negative quality control.

Further, positive quality control included pUC57 plasmid containing artificially synthesized Aspergillus 18S gene sequence at a concentration of 1X 10 ³ ~1×10 ⁶ pUC57 plasmid of sapiens/mL, artificially synthesized novel cryptococcus cap10 gene sequence, concentration of 1X 10 ³ ~1×10 ⁶ pUC57 plasmid of copies/mL artificially synthesized Yersinia pneumospore dhfr gene sequence at 1×10 concentration ³ ~1×10 ⁶ copies/mL, and pUC57 plasmid containing artificially synthesized GAPDH gene sequence at a concentration of 1X 10 ³ ~1×10 ⁵ copies/mL。

Further, the negative quality was controlled to pUC57 plasmid containing artificially synthesized GAPDH gene sequence at a concentration of 1X 10 ³ ~1×10 ⁵ copies/mL。

In another aspect, the invention provides the use of any one of the above combinations of primers and probes in the preparation of a multiplex quantitative PCR detection kit for Aspergillus, cryptococcus and Yersinia pneumospores.

All reagents employed in the present invention are commercially available.

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

the primer and probe combination and the kit for detecting aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumospore are prepared by designing the primer and the probe with specific sequence structures, are suitable for various detection samples, have high sensitivity and good specificity, have strong fluorescent signals, and provide reliable diagnosis guarantee for detecting clinical infection aiming at aspergillus, novel cryptococcus and yersinia pneumospore. Moreover, the aspergillus can cover various common aspergillus pathogens such as aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like, and has better broad spectrum.

Drawings

FIG. 1 is a graph of the results of example 4 with reference to Aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, cryptococcus neoformans, and Yersinia pneumospores.

FIG. 2 is a graph showing the results of detecting a negative sample in example 4.

FIG. 3 is a graph comparing 2500copies/mL Aspergillus, cryptococcus neoformans, and Yersinia pneumospore references detected in a comparative example using the combination of the primer and probe of example 1, respectively, with a control combination of the comparative example.

FIG. 4 is a graph comparing 2500copies/mL Aspergillus, cryptococcus neoformans, and Yersinia pneumospore references detected in a comparative example using the combination of the primer and probe of example 1, respectively, with a control combination of the comparative example.

FIG. 5 is a graph comparing 2500copies/mL Aspergillus, cryptococcus neoformans, and Yersinia pneumospore references, respectively, using the commercial products and the primer and probe combinations of example 1, respectively, in comparative examples.

FIG. 6 is a graph comparing 1000copies/mL Aspergillus, cryptococcus neoformans, and Yersinia pneumospore references, respectively, using the commercial products and the primer and probe combinations of example 1, respectively, in comparative examples.

Detailed Description

The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention. Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

Example 1 design of combinations of primers and probes for Aspergillus, cryptococcus neoformans, yersinia pneumospore detection

The 104 th-131 th site of the conserved DNA fragment in the aspergillus 18S gene is used as a detection target, the 104 th-131 th site of the conserved DNA fragment in the novel cryptococcus cap10 gene is used as a detection target, and the 104 th-131 th site of the conserved DNA fragment in the yersinia pneumospore Dhfr gene is used as a detection target. Primers were designed based on the target region sequences, and screened to obtain primers as shown in Table 1 below.

TABLE 1 nucleotide sequences of primers and probes of the examples of the invention

Wherein the 5 'end of SEQ ID No.3 marks ROX group, and the 3' end marks BHQ2 group; the 5 'end of SEQ ID No.6 marks FAM group, the 3' end marks BHQ2 group; the 5 'end of SEQ ID No.9 marks the CY5 group, and the 3' end marks the BHQ3 group.

Example 2 composition and preparation of real-time fluorescent quantitative PCR kit of Aspergillus, cryptococcus neoformans, yersinia pneumospores

The primer and probe combination, the PCR reaction MIX, the positive quality control and the negative quality control in the example 1 are adopted to prepare the detection kit.

PCR reaction MIX: comprises PCR buffer solution, dNTP, taq DNA polymerase and UDG enzyme. Wherein the PCR buffer solution is prepared from Tris-HCl (10-50 mM) and MgCl ₂ (3-6 mM), KCl (10-50 mM), triton X-100 (1-10%) and the like. Wherein dNTPs are a mixture of dATP (0.1-0.3 mM), dTTP (0.1-0.3 mM), dCTP (0.1-0.3 mM) and dGTP (0.1-0.3 mM), and the mixed solution further contains dUTP at 0.1-0.3 mM.

Wherein, the single detection PCR reaction MIX comprises 1.5U-3U Taq DNA polymerase and 0.5U-2U UDG enzyme.

The nature of yang is controlled: pUC57 plasmid containing artificially synthesized Aspergillus 18S gene sequence at concentration of 1×10 was included ³ ~1×10 ⁶ pUC57 plasmid of sapiens/mL, artificially synthesized novel cryptococcus cap10 gene sequence, concentration of 1X 10 ³ ~1×10 ⁶ pUC57 plasmid of copies/mL artificially synthesized Yersinia pneumospore dhfr gene sequence at 1×10 concentration ³ ~1×10 ⁶ copies/mL, and pUC57 plasmid containing artificially synthesized GAPDH gene sequence at a concentration of 1X 10 ³ ~1×10 ⁵ copies/mL。

Negative quality control: pUC57 plasmid containing artificially synthesized GAPDH gene sequence at concentration of 1×10 ³ ~1×10 ⁵ copies/mL。

The primers and probes of example 1 participated in the preparation reaction system, and the concentrations of each primer and probe are as follows: the final concentrations of the primer 18S-F1, the primer 18S-R5, the primer cap10-F3, the primer cap10-R2, the primer dhfr-F3 and the primer dhfr-R5 are respectively 0.1-0.5 mu M independently; the final concentrations of the probe 18S-P1-ROX, the probe cap10-P1-FAM and the probe dhfr-P1-CY5 are independently 0.05-0.25 mu M respectively.

Example 3 detection method of Aspergillus, cryptococcus neoformans, yersinia pneumospores

1. Sample source:

samples of different sources may be tested, and most samples are clinical samples, including but not limited to sputum samples, alveolar lavage samples, plasma samples, serum samples, cerebrospinal fluid samples, and the like.

2. Primer and probe:

primer and probe combinations for Aspergillus, cryptococcus neoformans, and Yersinia pneumospore detection in example 1 were used.

3. The experimental method comprises the following steps:

(1) Clinical samples were processed, and the processing procedure is exemplified for different samples as follows:

a) Sputum sample: and (3) adding the sputum sample into a 50mL test tube with a spiral cover, adding 1-2 times of 4% NaOH digestive juice by volume (about 3-5mL by volume of 1 time) into the sputum tube according to the viscosity of the sputum, screwing up the spiral cover, carrying out vortex oscillation for 1 minute, and placing the sputum in a biosafety cabinet for 15-20 minutes at room temperature to fully liquefy the sputum.

b) Alveolar lavage fluid sample: and (3) using Dithiothreitol (DTT) reagent, timely processing after receiving the sample, observing the property of the lavage fluid, for example, the sample contains a large amount of mucus, adding 2 times of 0.1g/dl DTT reagent, placing in a 37 ℃ water bath box, fully reversing and uniformly mixing every 10min, avoiding forced oscillation, and processing for 30 min-1 h, so that the sample mucus can be seen to be clarified.

c) Plasma samples: and (3) collecting 5mL of blood sample by using a vacuum blood collection tube containing EDTA anticoagulant, standing for 30 minutes at room temperature, centrifuging at 1500-2000 rpm for 10 minutes, and sucking the supernatant plasma into a sterile centrifuge tube.

d) Serum samples: and 5mL of blood sample is collected by using a vacuum negative pressure blood collection tube, standing is carried out at room temperature for 30 minutes, centrifugation is carried out at 1500-2000 rpm for 10 minutes, and supernatant plasma is sucked into a sterile centrifuge tube.

(2) Extracting and purifying nucleic acid from the clinical sample obtained in the step (1) by using a nucleic acid extraction reagent, wherein the specific steps of nucleic acid extraction are as follows:

taking a sample to be tested of not more than 200uL (adjusted according to the kit instructions), and extracting DNA in the sample to be tested according to instructions by using a Rhizopus genomic DNA/RNA extraction kit (product number DP 315) or QIAGEN-QIAamp DNA Mini Kit (product number 51304).

(3) And (3) preparation of a reagent:

each sample to be tested/positive quality control/negative quality control multiplex quantitative PCR reaction system comprises the following components: sample DNA/cation control/anion control (one of the three) 5 mu L, PCR reaction MIX 15 mu L, primer probe mixture 5 mu L, total reaction system 25 mu L.

Total test number n=number of samples to be tested (N) +number of positive quality controls (1) +number of negative quality controls (1). The amounts of the respective reagents added to the reaction system were calculated as shown in the following table 2:

TABLE 2 calculation of the amount of each reagent

Preparing a reaction system by using a 1.5 mL centrifuge tube (asepsis), adding all mixed solution of the MIX and the primer probe for PCR reaction, carrying out vortex oscillation for 10 seconds, centrifuging for standby, subpackaging 20 mu L/tube into the PCR reaction tube (asepsis and RNase-Free), and immediately putting the mixed solution of the MIX and the primer probe for PCR reaction into a temperature below-18 ℃ for freezing after the use.

(4) And (3) sample adding: and transferring the nucleic acid extracted from the sample to be detected, the positive quality control product and the negative quality control product to each PCR reaction tube by 5 mu L/tube respectively, covering a tube cover, centrifuging and transferring to a PCR detection area.

(5) And (3) PCR amplification: the PCR reaction tube was placed in a sample well of a fluorescent quantitative PCR instrument (ABI 7500). Setting a positive quality control product, a negative quality control product and a sample to be tested in sequence, and setting a sample name/number; defining a fluorescence detection channel: the ROX channel is an aspergillus nucleic acid detection channel, the FAM channel is a novel cryptococcus nucleic acid detection channel, the CY5 channel is a yarrowia pneumospore nucleic acid detection channel, and the VIC channel is an internal standard channel; the recommended PCR amplification procedure is shown in Table 3 below:

TABLE 3 PCR amplification procedure

(6) And (3) quality control:

controlling the nature of yang: FAM, ROX, CY5 and IC (internal standard) channels: ct is less than or equal to 35;

negative quality control: FAM, ROX, CY5 channel has no Ct value or Ct >40, VIC (internal standard) channel Ct <35;

the above requirements must be met simultaneously in one experiment. Otherwise, the experiment is deemed invalid and a retest is required.

(7) And (3) judging results:

the FAM channel detection has an obvious amplification curve, and the Ct value is less than or equal to 40, and the detection is judged to be positive to the novel cryptococcus;

the ROX channel detection has obvious amplification curve, and the Ct value is less than or equal to 40, and the detection is judged to be positive for aspergillus;

the CY5 channel is detected to have an obvious amplification curve, and the Ct value is less than or equal to 40, so that the Yersinia pneumoconiosis is judged to be positive;

FAM channel detection has no obvious amplification curve or has a detection Ct value of more than 40, VIC (internal standard) channel detection has obvious amplification curve and has a detection Ct value of less than or equal to 40, and the negative or concentration of aspergillus, cryptococcus neoformans and yersinia pneumospore is judged to be lower than the detection lower limit of the kit;

FAM channel detection has no obvious amplification curve or detection Ct value >40, VIC (internal standard) channel detection has no obvious amplification curve or detection Ct value >40, the test is invalid, and clinical resampling and repeated test are recommended.

The specific result judgment criteria are shown in Table 4:

TABLE 4 determination criteria for detection results

Example 4 detection of reference by Aspergillus, cryptococcus, yersinia pneumospore detection kit

The primers and probes shown in example 1 were used as references to the American Standard biological Collection (American Type Culture Collection, ATCC) in the manner described in example 2The detection after dilution to a specific concentration comprises: aspergillus fumigatus nucleic acid standard 1022DQ at 1E+05 copies/. Mu.L; aspergillus flavus nucleic acid standard, 9643DQ, at a concentration of 1E+05 copies/. Mu.L; aspergillus terreus nucleic acid standard, 20542DQ, at a concentration of 1E+05 copies/. Mu.L; aspergillus niger nucleic acid standard, 1015DQ, at 1E+05 copies/. Mu.L; yarrowia pneumospore nucleic acid standard, MYA-5006SD, at a concentration of 1E+05 copies/. Mu.L; novel cryptococcus nucleic acid standard, 66031D-5, 5 μg in mass. Meanwhile, taking a negative quality control product containing personnel internal references as a negative sample to be detected, wherein the concentration of the negative quality control product is about 1 multiplied by 10 ³ ~1×10 ⁵ copies/mL。

The detection results are shown in fig. 1 and 2. As can be seen from the results of FIG. 1, the primer and probe designed by the invention are adopted to detect six nucleic acid references, and experimental results meet expectations, so that the primer and probe combination of the invention can accurately identify aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumoconima; as can be seen from fig. 2, the detection result of the negative quality control product is negative. The ordinate of FIGS. 1 and 2 shows the relative fluorescence intensity (normalized dye fluorescence, father Rn).

Example 5 sensitivity test-detection sensitivity of Aspergillus, novel Cryptococcus, yersinia pneumospore detection kit

The above six nucleic acid references were each serially diluted to 2500copies/mL, 1000copies/mL, 500copies/mL, 250 copies/mL as test samples using simulated sputum, serum, plasma negative samples, each gradient was repeated 20 times, each with three batches of the composition, and each with 95% detection level as the lowest limit of detection, as shown in tables 5-13 below:

table 5 simulation of detection limits of three reagent batches for sputum samples (first batch)

Table 6 simulation of the results of three reagent runs (second run) on sputum samples

Table 7 simulation of the results of three reagent runs (third run) on sputum samples

TABLE 8 detection limits for three reagent batches of serum samples (first batch)

TABLE 9 detection limits of three reagent batches for serum samples (second batch)

TABLE 10 detection limit of serum sample three-batch reagent (third batch)

TABLE 11 detection limits for three batches of reagent for plasma samples (first batch)

TABLE 12 detection limits for three batches of reagent for plasma samples (second batch)

TABLE 13 detection limits of three reagent batches for plasma samples (third batch)

Table 14 statistics of test results for three samples to be tested

As can be seen from the results shown in tables 5 to 13, the three samples to be tested (sputum, serum and plasma) are respectively detected for 20 times by the combination of three batches of primers and probes, and the positive detection rate is over 95% when the positive sample concentration is 500copies/mL, so that the sensitivity is high. Therefore, the primer probe combination reagent has higher detection sensitivity to aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumospore, and can be used as a kit for detecting nucleic acid of aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumospore.

Example 6 specificity and interference of Aspergillus, cryptococcus, yersinia pneumospore detection kits

1. Specificity: samples of 20 pathogens which have homology with aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus and aspergillus niger), cryptococcus neoformans and yersinia pneumospore detection kits and are easy to cause the same or similar clinical symptoms are used as interfering object samples, and the information of the samples and the detection results are shown in tables 15-16.

TABLE 15 approximate pathogen and its concentration

Approximating pathogens	Concentration of
		Candida albicans	1.00E+06copies/mL
Candida tropicalis	1.00E+06copies/mL
		Candida glabrata (C.glabrata)	1.00E+06copies/mL
Candida parapsilosis	1.00E+06copies/mL
		Candida krusei (Fr.) Kuntze	1.00E+06copies/mL
Penicillium marneffei	1.00E+06copies/mL
		Pneumosporium californicum (L.) pers	1.00E+06copies/mL
Staphylococcus aureus	1.00E+06copies/mL
		Streptococcus pneumoniae	1.00E+06copies/mL
Haemophilus haemolyticus	1.00E+06copies/mL
		Coli bacterium	1.00E+06copies/mL
Acinetobacter baumannii	1.00E+06copies/mL
		Moraxella catarrhalis	1.00E+06copies/mL
Pseudomonas aeruginosa	1.00E+06copies/mL
		Klebsiella pneumoniae	1.00E+06copies/mL
Chlamydia pneumoniae	1.00E+06copies/mL
		Influenza A virus	1.00E+06copies/mL
Influenza b virus	1.00E+06copies/mL
		Parainfluenza virus	1.00E+06copies/mL
Respiratory syncytial virus	1.00E+06copies/mL

TABLE 16 detection results of interferent samples

The above 20 interferents were mixed with Aspergillus (including Aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, etc.), cryptococcus neoformans, and Yersinia pneumospore references at the lowest limit of detection (LOD) concentration to obtain samples, and the samples were tested using different batches of kits, and the test results are shown in Table 17.

Table 17 results of three reagent lot competitive interference assays

As can be seen from the test results in Table 16, the specificity of 20 pathogens was measured by the three batches of the test agents of the present invention, which showed no negative and no false positive.

As can be seen from Table 17, after mixing 20 pathogen interferents with a LOD concentration of Aspergillus (including Aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, etc.), cryptococcus neoformans, and Yersinia pneumospore references, the three test reagents of the present invention were successful in detecting a LOD concentration reference. Therefore, the invention can eliminate the interference of different pathogens and accurately detect the carrying condition of aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumoconica.

2. Interference: the statistics of each interferent and the concentration thereof are shown in table 18, pathogen nucleic acid references with LOD concentration added to each interferent are used as samples to be tested, the samples to be tested are detected by using three batches of kits, and the detection results are shown in table 19.

Table 18, the addition of interfering substances and concentration

TABLE 19 detection results of interferents

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As can be seen from Table 19, the invention can accurately detect Aspergillus species (including Aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger, etc.), novel cryptococcus, and Yersinia pneumospore reference species with LOD concentration in the presence of exogenous interferents at different concentrations, and Ct value is not significantly different from that of the control.

In summary, under the condition that different interferents exist, the invention can accurately detect the existence of aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumoconima in a sample to be detected.

Therefore, the invention has strong anti-interference capability on various interferents and good specificity on aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus, aspergillus niger and the like), novel cryptococcus and yersinia pneumoconica.

Comparative example A reference sample of Aspergillus, cryptococcus neoformans and Yersinia pneumospore was detected using the probe primer combination of example 1 and the control combination, respectively

In the use of nucleic acid detection platforms, the combination of primers and probes is a critical factor affecting the detection capacity. Under different detection conditions, such as in the presence of multiple pathogens or different interferents, the combination of primers and probes used is required to maintain high specificity and sensitivity, and simultaneously has good amplification curve and detection signal intensity. The combination of the candidate primer and the probe designed by the applicant is compared (see table 20 for details), namely a control combination I and a control combination II respectively, and aspergillus (including aspergillus fumigatus, aspergillus flavus, aspergillus terreus and aspergillus niger), novel cryptococcus and yersinia pneumospore references are used as samples to be tested, and the detection is carried out by using an ABI 7500 platform, wherein the results are shown in figures 3-4, and the ordinate of figures 3 and 4 are relative fluorescence intensity (normalized dye fluorescence, rn). Wherein the concentration of Aspergillus (including Aspergillus fumigatus, aspergillus flavus, aspergillus terreus and Aspergillus niger), novel Cryptococcus and Yersinia pneumospore reference substances in the sample to be detected is 2500 copies/mL.

TABLE 20 nucleotide sequences of primers and probes of comparative examples of the present invention

From the results of FIGS. 3-4, it can be seen that the Ct values of the first control and the second control are generally lagged by about 2-4 compared with the present invention at the same concentration of Aspergillus, cryptococcus neoformans and Yersinia pneumospore templates, and that the primer and probe combinations of the present invention have not only lower Ct but also fluorescence signal values about 2 times that of the alternative combinations. Thus, the amplification efficiency of the combination of the primer and probe of the present application is higher.

The combination of the primer and the probe of the invention is compared with commercial products, the sample to be detected is 2500copies/mL and 1000copies/mL Aspergillus (including Aspergillus fumigatus, aspergillus flavus, aspergillus terreus and Aspergillus niger), novel cryptococcus and Yersinia pneumospore reference, and the detection result is shown in Table 21 and figures 5-6, and the ordinate of figures 5 and 6 is relative fluorescence intensity (normalized dye fluorescence, # Rn) by using an ABI 7500 platform.

TABLE 21 detection Ct values for the combination and comparison kit of the invention

As can be seen from Table 21 and FIGS. 5 to 6, the Ct value of the combination of the primer and the probe of the present invention is smaller than that of the commercially available product and the fluorescence intensity is high at a template concentration of 2500copies/mL (FIG. 5); the combination of the primer and probe of the present invention was effectively detected at a template concentration of 1000copies/mL, but the commercially available product had no response value (FIG. 6). Therefore, the combination of the primer and the probe of the invention can effectively avoid missed detection when detecting a low-concentration sample.

The present invention has been disclosed in the preferred embodiments, but it is not limited thereto, and the equivalent substitution or equivalent transformation can be adopted to achieve the technical solution within the scope of the present invention.

Claims

1. A combination of primers and probes for multiplex quantitative PCR detection of aspergillus, cryptococcus neoformans and yersinia pneumospores, comprising:

2. The primer and probe combination of claim 1, wherein: the 5 'end of the probe is marked with a fluorescence report group, and the 3' end of the probe is marked with a fluorescence quenching group; wherein the fluorescent reporter group is selected from any one of FAM, VIC, HEX, CY, CY3, JOE and ROX; the fluorescence quenching group is selected from any one of BHQ1, BHQ2, BHQ3 and TAMRA.

3. The primer and probe combination of claim 2, wherein: a fluorescent group ROX is marked at the 5 'end of the nucleotide sequence shown in SEQ ID No.3, and a quenching group BHQ2 is marked at the 3' end; marking a fluorescent group FAM at the 5' -end of the probe with the nucleotide sequence shown in SEQ ID No. 6; 3' end marks quenching group BHQ2; the 5 '-end of the probe of the nucleotide sequence shown in SEQ ID No.9 is marked with a fluorescent group CY5, and the 3' -end is marked with a quenching group BHQ3.

4. Kit for multiplex quantitative PCR detection of aspergillus, cryptococcus neoformans, yersinia, comprising a combination of a primer and a probe according to any one of claims 1-3.

5. The kit according to claim 4, wherein the final concentrations of the primer 18S-F1, the primer 18S-R5, the primer cap10-F3, the primer cap10-R2, the primer dhfr-F3 and the primer dhfr-R5 are independently 0.1-0.5. Mu.M; the final concentrations of the probe 18S-P1-ROX, the probe cap10-P1-FAM and the probe dhfr-P1-CY5 are independently 0.05-0.25 mu M respectively.

6. The kit of claim 4, wherein: the kit also comprises an amplification reaction liquid mixture, wherein the amplification reaction liquid mixture comprises a PCR buffer solution, a dNTP solution, taq DNA polymerase and UDG enzyme.

7. The kit of claim 6, wherein: the PCR buffer solution comprises Tris-HCl and MgCl ₂ KCl and Triton X-100, tris-HCl concentration of 10-50 mM in PCR buffer ₂ The concentration of (2) is 3-6 mM, the concentration of KCl is 10-50 mM, and the concentration of Triton X-100 is 1% -10%; the dNTP solution comprises dATP, dTTP, dCTP, dGTP and dUTP, wherein the dATP concentration in the dNTP solution is 0.1-0.3 mM, the dTTP concentration in the dNTP solution is 0.1-0.3 mM, the dCTP concentration in the dCTP solution is 0.1-0.3 mM, the dGTP concentration in the dGTP solution is 0.1-0.3 mM, and the dUTP concentration in the dNTP solution is 0.1-0.3 mM; the single detection reaction liquid contains 1.5U-3U of TaqDNA polymerase and 0.5U-2U of UDG enzyme.

8. The kit of claim 4, wherein: also includes positive quality control and negative quality control.

9. The kit of claim 8, wherein: cationic control included pUC57 plasmid containing artificially synthesized Aspergillus 18S gene sequence at a concentration of 1X 10 ³ ~1×10 ⁶ pUC57 plasmid of sapiens/mL, artificially synthesized novel cryptococcus cap10 gene sequence, concentration of 1X 10 ³ ~1×10 ⁶ pUC57 plasmid of copies/mL artificially synthesized Yersinia pneumospore dhfr gene sequence at 1×10 concentration ³ ~1×10 ⁶ copies/mL, and pUC57 plasmid containing artificially synthesized GAPDH gene sequence at a concentration of 1X 10 ³ ~1×10 ⁵ cobies/mL; the negative property was controlled to be pUC57 plasmid containing artificially synthesized GAPDH gene sequence at a concentration of 1X 10 ³ ~1×10 ⁵ copies/mL。

10. Use of a combination of primers and probes according to any one of claims 1-3 for the preparation of a multiplex quantitative PCR detection kit for aspergillus, cryptococcus neoformans, yersinia pneumospores.