A method and kit for detecting fungi using multiplex quantitative PCR

Abstract

This invention discloses a method for simultaneously identifying fungi using multiplex quantitative PCR technology. Specific primers and probes are designed for four important fungi: Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jirovecii, and Rhizomucor pusillus. Using multiplex fluorescence quantitative PCR, the method simultaneously identifies clinical samples for infection with these three fungi, reducing the detection cycle and cost of these important fungi, and effectively enabling rapid diagnosis of fungal infections.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biological detection technology and molecular diagnosis, and in particular to a method for simultaneously identifying four important fungi using multiplex quantitative PCR technology. Background Art

[0002] Most fungi are ubiquitous in the environment, and humans are exposed to them through inhalation of spores or small yeast cells. Over the past few decades, the clinical relevance of fungal diseases has increased significantly due to the increasing number of immunocompromised patients. According to incomplete estimates, approximately 71 million people in my country suffer from fungal diseases. In addition to HIV / AIDS or hematologic malignancies, a variety of emerging host risk factors, particularly COPD, asthma, and lung cancer, have been associated with fungal diseases. Previous studies have shown that the mortality rate of opportunistic fungal infections exceeds 50%, and in bone marrow transplant recipients infected with Aspergillus, the mortality rate is as high as 95%. Common fungal diseases include aspergillosis, mucormycosis, and pulmonary cryptococcosis.

[0003] Aspergillus fumigatus is the leading cause of human aspergillosis. Globally, there are approximately 4.8 million cases of allergic bronchopulmonary aspergillosis, 3 million cases of chronic pulmonary aspergillosis, and 250,000 cases of invasive aspergillosis each year. Furthermore, A. fumigatus has become one of the most common causes of infectious death in severely immunocompromised patients. In recent years, the incidence of mucormycosis has been increasing globally, with particularly high rates among patients with uncontrolled diabetes in India and China. Mucormycosis progresses rapidly and is difficult to diagnose, primarily affecting immunocompromised patients and those with diabetes. Many cases remain undiagnosed due to the difficulty in obtaining specimens from deep tissues and the low sensitivity of diagnostic tests. Mucormycosis, caused by Rhizomucor microsporus, is typically found in the lungs of patients with weakened immune systems and often has fatal consequences. Cryptococcus neoformans is a ubiquitous opportunistic yeast. Cryptococcus neoformans is a widespread, disseminated infection worldwide, and the vast majority of patients with symptomatic, disseminated infection have an underlying immunocompromised condition. Most Cryptococcus neoformans infections occur in the lungs, but fungal meningitis, particularly as a secondary infection in HIV patients, is often caused by Cryptococcus neoformans. Furthermore, with the widespread use of mNGS in recent years, the diagnosis of Pneumocystis jirovecii pneumonia, caused by Pneumocystis jirovecii, has increased. It is a conditional lung infection. Given the rapid progression and high mortality rate of Pneumocystis jirovecii pneumonia, rapid identification of Pneumocystis jirovecii facilitates timely anti-infective treatment and improves prognosis.

[0004] At present, clinical fungal detection technologies include morphological examination, culture, identification, drug sensitivity testing, immunological testing, etc. However, these methods either take a long time to culture or have low specificity. Pneumocystis jiroveci is an atypical fungus and cannot be detected by culture on a culture medium. Molecular biology technology has the characteristics of high detection specificity, simple and convenient operation, and low price, and has certain applications in fungal detection. However, since traditional qPCR can only detect a single species, repeated operations are required when detecting multiple species. Not only is the experimental process time-consuming, but the detection cost is also high, which cannot meet the needs of clinical rapid diagnosis of pathogens. Therefore, it is very important to develop a method that can quickly identify a variety of common fungi. Summary of the Invention

[0005] The present invention provides a method for simultaneously identifying four common fungi using multiplex quantitative PCR technology. It can simultaneously detect four important fungi, Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jirovecii and Rhizomucor pusillus, at one time. It has the advantages of high sensitivity, strong specificity, short detection cycle, high efficiency and low cost, and reflects higher feasibility and application prospects.

[0006] A kit for multiplex quantitative PCR detection of fungi, comprising a primer combination for multiplex quantitative PCR reaction to detect fungi, the primer combination comprising:

[0007] The forward and reverse primer pairs used for detecting Aspergillus fumigatus, the nucleotide sequences of which are shown in SEQ ID NO.1-2;

[0008] The forward and reverse primer pairs used for detecting Cryptococcus neoformans, the nucleotide sequences of which are shown in SEQ ID NO.4-5;

[0009] The forward and reverse primer pairs for detecting Pneumocystis jiroveci and Aspergillus fumigatus have nucleotide sequences as shown in SEQ ID NOs. 7-8;

[0010] The forward and reverse primer pairs used for detecting Rhizomucor pusillus have nucleotide sequences shown in SEQ ID NOs. 10-11.

[0011] The kit also includes a probe combination for reporting the amplification products; the probe combination includes:

[0012] A probe for reporting the amplified product of Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ ID NO. 3;

[0013] A probe for reporting the amplified product of Cryptococcus neoformans, the nucleotide sequence of which is shown in SEQ ID NO.6;

[0014] A probe for reporting the amplified product of Pneumocystis jirovecii, the nucleotide sequence of which is shown in SEQ ID NO.9;

[0015] The nucleotide sequence of the probe used to report the amplified product of Rhizomucor pusillus is shown in SEQ ID NO.12.

[0016] The 5' end of the probe is modified with a reporter group, which is FAM, HEX, ROX and Cy5; the four probes are modified with different reporter groups.

[0017] The 3' end of the probe is modified with a quenching group, which is BHQ1 and BHQ2; the nucleotide sequence shown in SEQ ID NO.3 and the nucleotide sequence shown in SEQ ID NO.6 are modified with the quenching group BHQ1, and the nucleotide sequence shown in SEQ ID NO.9 and the nucleotide sequence shown in SEQ ID NO.12 are modified with the quenching group BHQ2.

[0018] A method for detecting fungi using multiplex quantitative PCR, which uses the above-mentioned kit to perform multiplex quantitative PCR reactions to identify Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jirovecii and Rhizomucor pusillus.

[0019] The method further includes the step of performing multiple PCR reactions on the samples.

[0020] The samples include but are not limited to alveolar lavage fluid, sputum, blood, cerebrospinal fluid, pericardial effusion, pleural effusion, urine, pus, swabs and tissues.

[0021] The reagents in the multiplex quantitative PCR reaction include: 2×Hieff 12.5 μL of Universal TaqMan multiplex qPCR master mix, the final concentration of Primer Mix (10 μM) is 0.2 μM, the final concentration of Probe Mix (10 μM) is 0.1 μM, 1 μL of 10 ng / μL DNA template, and 25 μL of enzyme-free water are added.

[0022] The reaction procedure of the multiplex quantitative PCR amplification was as follows: pre-denaturation at 95° C. for 5 min; denaturation at 95° C. for 15 s; annealing at 58.5° C. for 30 s; and amplification for 40 cycles.

[0023] In the described method, if the FAM fluorescent modified probe has an obvious S-shaped amplification curve, it indicates that Aspergillus fumigatus is present in the sample to be tested; if the HEX fluorescent modified probe has an amplification curve and satisfies Ct < 40, it indicates that Cryptococcus neoformans is present in the sample to be tested; if the ROX fluorescent modified probe has an obvious S-shaped amplification curve, it indicates that Pneumocystis jirovecii is present in the sample to be tested; if the Cy5 fluorescent modified probe has an obvious S-shaped amplification curve, it indicates that Rhizomucor microsporus is present in the sample to be tested.

[0024] Beneficial effects

[0025] The present invention utilizes multiplex quantitative PCR technology to simultaneously identify four important fungi. The detection primer set is highly specific and cannot be matched to sequences from other pathogens. This method is highly operational and can simultaneously identify four important fungi—Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jiroveci, and Rhizomucor pusillus—in the same reaction system. This significantly simplifies the operational complexity of detection, effectively shortens the time and cost of clinical testing for these four important fungi, and significantly improves the efficiency of clinical pathogen detection, thus possessing strong application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 When the FAM, HEX, ROX, and Cy5 fluorescent modified probes all have amplification curves and the Ct of HEX is less than 40, it indicates that the four fungi, Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jirovecii, and Rhizomucor pusillus, are present in the test sample;

[0027] Figure 2 Figure 2 is a graph of the sensitivity amplification curve of specific probes. A is Aspergillus fumigatus, B is Cryptococcus neoformans, C is Pneumocystis jiroveci, and D is Rhizomucor pusillus; (2860, 286, 29, 14, 7, 4, 0 copies, NTC)

[0028] Figure 3 Amplification curve results for specificity verification

[0029] Figure 4 This is the test result of clinical positive samples

[0030] Figure 5 This is the test result of clinical negative samples DETAILED DESCRIPTION

[0031] In order to facilitate a better understanding of the present invention, the present invention will be further described below in conjunction with relevant drawings and embodiments. It should be noted that the following description is only for further explanation of the present invention and does not limit its content.

[0032] The experimental materials, reagents and instruments used in the present invention are as follows:

[0033] Experimental materials: Positive plasmids of Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jiroveci and Rhizomucor parvum, and blood or body fluid samples from clinically infected patients.

[0034] Experimental reagents: magnetic bead-based large-volume free nucleic acid extraction kit (Tiangen: DP710-T2 or QIAamp Circulating Nucleic Acid Kit (50): 55114); probe and primer synthesis (Nanjing GenScript Biotechnology Co., Ltd.); Hieff Universal TaqMan multiplex qPCR master mix (Yisheng: 11211ES08).

[0035] Experimental instruments: Fluorescence quantitative PCR instrument (Bio-Rad: CFX384); microplate mini centrifuge (Qilin Bell: BE-6100).

[0036] Example 1

[0037] Design and optimization of specific primers and probes

[0038] 1. Probe primer design

[0039] Download the complete genome sequence of the species, identify its consensus sequence, and then apply a sliding window across the genome sequence. Using the parameters of a primer design template window length of 100-500bp and a window sliding length of 10-50bp, generate candidate primer design template sequences for the species. These sequences are then compared with genome sequences from other species to assess the specificity of the primer design template sequences. Furthermore, basic primer design principles must be met: a GC content between 30-60%; primers F and R have the same annealing temperature of approximately 60°C, while primer P must anneal at approximately 65°C; and primers must be free of primer dimers.

[0040] Based on the above screening principles, we screened Aspergillus fumigatus, Cryptococcus Neoformans, Pneumocystis jirovecii, and Rhizomucor pusillus-specific gene sequences as templates for primer design. The nucleotide sequences of the designed primers and probes are shown in Table 1.

[0041] Table 1 Nucleotide sequences of primers and probes

[0042]

[0043] 2. Positive plasmid synthesis

[0044] The PCR product sequences of Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jiroveci and Rhizomucor pusillus were cloned into the pUC57 vector to obtain four fungus-positive plasmids. The inserted nucleotide sequence is shown in SEQ ID NO.13:

[0045]

[0046] 3. Preparation of Standards

[0047] The synthetic plasmid concentration was diluted to 1 ng / μL, the fragment size was 3546 bp, and the copy number was (6.02×10 23 )×1ng / μL×10 -9 ) / (3546bp×660)=2.57×10 8 The plasmid was diluted 10-fold to 2.57 × 10 5 copies / μL, and the plasmid synthesized at this concentration was used as a template for multiplex fluorescence quantitative PCR detection.

[0048] 4. Multiplex Fluorescence Quantitative PCR Detection

[0049] Prepare the qPCR reaction system according to Table 2:

[0050] Table 2 qPCR reaction system

[0051]

[0052]

[0053] Set up the qPCR reaction program according to Table 3 and collect fluorescence during the annealing / extension step.

[0054] Table 3 qPCR reaction procedure

[0055]

[0056] Result analysis: Multiplex fluorescence quantitative PCR amplification curve is as follows: Figure 1 The graph shows the relationship between the intensity of the four fluorescence and the number of amplification cycles. All four targets have S-shaped amplification curves, and the detection effect is best when the annealing temperature is 58.5℃. Figure 1 .

[0057] Example 2

[0058] Multiplex real-time fluorescence method validation

[0059] 1. Sensitivity Verification

[0060] (1) Preparation of standard products

[0061] Take 2.57×105 The plasmid with a concentration of 10 copies / μL was diluted 10-fold to obtain positive synthetic plasmids with concentrations of 2570, 257, 26, 13, 7, and 4 copies / μL as DNA templates. 1 μL of template was added to each reaction well, and 3 replicates were set for each gradient.

[0062] (2) Multiplex fluorescence quantitative PCR detection

[0063] The detection primers and detection probes for Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jiroveci and Rhizomucor pusillus in step 1 of Example 1 were used as primers and probes, and multiplex fluorescence quantitative PCR detection was performed according to step 4 of Example 1. The results are shown in FIG. Figure 2 As shown in Figure 2, the amplification curves of 2570, 257, 26, 13, and 7 copies are S-shaped, and all three replicates can be detected. Therefore, the detection limit of the four fungal multiplex fluorescence quantitative PCR of the present invention is 7 copies / reaction. The sensitivity verification amplification curve results of the four species are shown in Figure 2. Figure 2 .

[0064] 2. Specificity Verification

[0065] (1) Sample DNA extraction

[0066] DNA was extracted from body fluid samples using a large-volume magnetic bead-based free nucleic acid extraction kit or QIAamp Circulating Nucleic Acid Kit. For specific steps, refer to the kit instructions.

[0067] (2) Multiplex fluorescence quantitative PCR detection

[0068] The specificity of the Aspergillus fumigatus probe primers was verified using mNGS positive samples of Aspergillus fumigatus and Aspergillus oryzae; the specificity of Cryptococcus neoformans was verified using mNGS positive samples of Cryptococcus gattii VGII type; the specificity of Rhizomucor microti was verified using mNGS positive samples of Mucor miei; Pneumocystis jirovecii had no annotated cognate bacteria, so this test was not performed. The detection primers and detection probes for Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jirovecii and Rhizomucor microti in step 1 of Example 1 were used as primers and probes, and multiplex fluorescence quantitative PCR detection was performed according to step 4 of Example 1. The results are shown in Tables 4 and Figure 3 The results showed that the probe primers designed by the present invention have strong specificity.

[0069] Table 4 Specificity verification experimental results

[0070]

[0071] Example 3

[0072] Clinical sample testing

[0073] 1. Sample DNA Extraction

[0074] The multiplex real-time fluorescence PCR method developed by this invention, which simultaneously detects Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jiroveci, and Rhizomucor parvum, was used to test 12 samples from clinically infected patients, including bronchoalveolar lavage fluid and blood. DNA was extracted from body fluid samples using either a magnetic bead-based large-volume cell-free nucleic acid extraction kit or the QIAamp Circulating Nucleic Acid Kit. Specific procedures are described in the kit instructions.

[0075] 2. Multiplex fluorescence quantitative PCR detection

[0076] The detection primers and detection probes for Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jiroveci and Rhizomucor pusillus in step 1 of Example 1 were used as primers and probes, and multiplex fluorescence quantitative PCR detection was performed according to step 4 of Example 1. The results are shown in Table 5, which show that the method established by the present invention is completely consistent with the mNGS sequencing results, and the present invention is accurate and reliable. The detection results of some clinical positive samples and negative samples are shown in Table 5. Figure 4 and Figure 5 .

[0077] Table 5 Clinical sample test results Fluorescence quantitative PCR detection Ct value

[0078]

[0079] The above-described embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that improvements and modifications may be made by those skilled in the art without departing from the principles of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A kit for multiplex quantitative PCR detection of fungi, characterized in that: The primer combination is used for multiplex quantitative PCR reaction to detect fungi, and the primer combination includes: The forward and reverse primer pairs used for detecting Aspergillus fumigatus, the nucleotide sequences of which are shown in SEQ ID NO.1-2; The forward and reverse primer pairs for detecting Cryptococcus neoformans have nucleotide sequences shown in SEQ ID NOs. 4-5; the forward and reverse primer pairs for detecting Pneumocystis jiroveci and Aspergillus fumigatus have nucleotide sequences shown in SEQ ID NOs. 7-8; Forward and reverse primer pairs for detecting Rhizomucor microti, the nucleotide sequences of which are shown in SEQ ID NOs. 10-11; the kit also includes a probe combination for reporting the amplified products; the probe combination includes: a probe for reporting the amplified product of Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ ID NO. 3; A probe for reporting the amplified product of Cryptococcus neoformans, the nucleotide sequence of which is shown in SEQ ID NO.6; A probe for reporting the amplified product of Pneumocystis jirovecii, the nucleotide sequence of which is shown in SEQ ID NO.9; For reporting the amplified product of Rhizomucor pusillus, the nucleotide sequence of the probe is shown in SEQ ID NO.12; The 5' end of the probe is modified with a reporter group, the reporter group is FAM, HEX, ROX and Cy5; four probes are modified with different reporter groups; The 3' end of the probe is modified with a quencher group, the quencher group is BHQ1 and BHQ2; The nucleotide sequence shown in SEQ ID NO.3 and the nucleotide sequence shown in SEQ ID NO.6 were modified with the quenching group BHQ1, and the nucleotide sequence shown in SEQ ID NO.9 and the nucleotide sequence shown in SEQ ID NO.12 were modified with the quenching group BHQ2.

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