A primer probe set, kit and detection method for multiplex qPCR detection of pathogens

Abstract

The present invention provides a multiplex PCR primer, probe and detection method for detecting common clinical pathogens. The present invention analyzes and compares the gene sequences of common clinical pathogens Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis and Mycobacterium abscessus, designs fluorescent PCR detection primers and probes, and simultaneously identifies whether clinical samples are infected with these four pathogens at one time. The detection method of the present invention simplifies the original detection workload, shortens the detection time, and provides an etiological basis for clinical diagnosis and treatment, and plays an important guiding role in the targeted selection of antibiotics for clinical use.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and medicine, and specifically relates to a multiplex PCR primer, a probe and a detection method for detecting common clinical pathogens. Background Art

[0002] It is estimated that about 1 billion people are infected with fungi worldwide, the number of patients with serious fungal diseases exceeds 150 million, and the number of deaths caused by fungal diseases is about 1.6 million cases per year. Invasive aspergillosis mainly occurs in hosts with severely impaired immune function.

[0003] Staphylococcus aureus is an important zoonotic pathogen that can cause a variety of infections and diseases in humans and animals, including skin infections, pneumonia, infectious arthritis, endocarditis, sepsis, etc. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important pathogens causing hospital-associated and community-associated infections, and its morbidity and mortality rates have remained high, posing a huge threat to clinical practice.

[0004] According to estimates by the World Health Organization, about a quarter of the world's population is infected with Mycobacterium tuberculosis and remains in a state of latent infection with Mycobacterium tuberculosis for a long time. In the absence of preventive treatment, about 5%-10% of people with latent infection with Mycobacterium tuberculosis will develop active tuberculosis.

[0005] For the above four pathogens, the most common clinical detection methods are mostly isolation and culture, but this method has a long detection cycle, low sensitivity and poor specificity. Molecular diagnostic technology can effectively make up for the shortcomings of traditional methods. Fluorescence quantitative PCR technology introduces fluorescent labeled probes into the PCR reaction system to achieve its quantitative function. Fluorescence quantitative PCR technology has been maturely applied in pathogen detection, but the above four pathogens are mostly detected separately, and the specific methods, amplification reagents and detection conditions used for each detection are different, which greatly increases the workload and cost of clinical detection. Therefore, it is urgent to establish a rapid, sensitive and specific detection technology to improve the clinical detection ability of Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis and Mycobacterium abscessus. Summary of the invention

[0006] The purpose of the present invention is to use a multiplex fluorescence PCR method to provide primers, probes and a detection method for simultaneously detecting four pathogenic microorganisms, namely, Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis and Mycobacterium abscessus, in clinical infection specimens. The method has the advantages of high sensitivity, strong specificity, short detection cycle, high efficiency and low cost, and embodies higher feasibility and application prospects.

[0007] The present invention first provides a primer probe set for multiplex qPCR detection of pathogens, which comprises a quadruple fluorescent PCR primer combination suitable for simultaneously detecting Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, and Mycobacterium abscessus in a single reaction;

[0008] The primer combination comprises:

[0009] A forward and reverse primer pair for detecting Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ ID NO.1-2;

[0010] A forward and reverse primer pair for detecting Staphylococcus aureus, the nucleotide sequence of which is shown in SEQ ID NO.4-5;

[0011] A forward and reverse primer pair for detecting Mycobacterium tuberculosis, the nucleotide sequence of which is shown in SEQ ID NO.7-8;

[0012] A forward and reverse primer pair for detecting Mycobacterium abscessus, the nucleotide sequences of which are shown in SEQ ID NOs. 10-11;

[0013] The probe combination includes:

[0014] A probe for reporting the amplified product of Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ ID NO.3;

[0015] A probe for reporting the amplified product of Staphylococcus aureus, the nucleotide sequence of which is shown in SEQ ID NO.6;

[0016] A probe for reporting the amplified product of Mycobacterium tuberculosis, the nucleotide sequence of which is shown in SEQ ID NO.9;

[0017] The probe used to report the amplified product of Mycobacterium abscessus, the nucleotide sequence of which is shown in SEQ ID NO.12.

[0018] The 5' end of the probe is modified with a reporter group, and the reporter group is FAM, HEX, ROX and Cy5; the four probes are modified with different reporter groups.

[0019] 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.

[0020] A kit comprises the above-mentioned primer probe set.

[0021] A multiplex qPCR method for detecting pathogens, wherein the primer probe set is used to perform a quadruple fluorescent PCR reaction and determine the sample; the detection method is used for simultaneously detecting Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, and Mycobacterium abscessus in a single reaction.

[0022] The samples include but are not limited to alveolar lavage fluid, sputum, blood, cerebrospinal fluid, pericardial effusion, pleural effusion, urine, pus, swabs and tissues.

[0023] The multiplex fluorescence quantitative PCR detection method comprises the following steps: extracting genomic DNA of the sample as a template, using the above-mentioned detection primers and detection probes to perform multiplex fluorescence quantitative PCR amplification, after the reaction is completed, reading and recording the PCR amplification cycle number Ct of the sample according to the fluorescence signal of the fluorescent reporter group labeled with the probe, and judging whether the sample contains the above-mentioned four pathogenic microorganisms according to the Ct value of each sample and the established judgment standard.

[0024] The multiplex fluorescence PCR detection system is: 2×Hieff Unicon ® 5 μL of Universal TaqMan multiplex qPCR master mix, 1.2 μL of four probe primer premixes, with final concentrations of 10 μM respectively, and 3.8 μL of DNA template.

[0025] The multiplex fluorescence quantitative PCR amplification program is preferably: 95°C for 5 minutes; 95°C for 15 seconds, 58.5°C for 30 seconds, and 40 cycles.

[0026] During the sample determination, negative and positive controls were set up, and the results were determined based on the Ct value of the amplification curve.

[0027] The positive control should have a clear S-shaped amplification curve, with good reproducibility of the three replicate wells and Ct values ​​of <30; at least two of the three replicate wells of the negative control should have no Ct value and no typical S-shaped curve.

[0028] The samples are being judged, and the judgment criteria are as follows:

[0029] If the sample well has a Ct value and an obvious S-shaped amplification curve, it means that the corresponding bacteria of the sample is positive. If the sample well has no Ct value and an obvious S-shaped amplification curve, it means that the corresponding bacteria of the sample is negative. Beneficial Effects

[0030] The present invention designs multiple specific primers and probes for the conserved regions of the specific genes of four common human pathogenic microorganisms. Through laboratory condition optimization and screening, a method for simultaneously detecting these four pathogens in a single reaction was established, which simplified the original detection workload. There was no significant difference between the quadruple qPCR detection and the single-reaction detection, and the detection sensitivity of clinical positive samples was high, and there was no nonspecific amplification of clinical negative samples, showing good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1-Figure 4 They are respectively the fluorescence PCR amplification curves of the primer-probe combinations designed for four pathogens, namely, Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, and Mycobacterium abscessus.

[0032] Figure 5-Figure 6 These are the fluorescence PCR amplification curves of the negative control and the positive control, respectively.

[0033] Figure 7-Figure 8 They are the amplification curves of multiplex fluorescence PCR and single fluorescence PCR respectively. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following description is only for explaining the present invention and does not limit its contents.

[0035] The reagents and instruments used in the present invention are as follows:

[0036] Reagents used: Hieff Unicon® Universal TaqMan multiplex qPCR master mix: YEASEN; 11211ES08

[0037] Probes for Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, and Mycobacterium abscessus

[0038] Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, Mycobacterium abscessus upstream and downstream primer working solution

[0039] Positive Control

[0040] Negative Control

[0041] Instrument used: CFX 384 Optics Module: Bio-Rad; CFX384

[0042] Gilson pipette; Gilson

[0043] Scilogex handheld centrifuge: Scilogex; S1010E

[0044] IKA Vortex 2: IKA; V2 SO25

[0045] Microplate mini centrifuge: Qilin Bell; BE-6100

[0046] Hard-Shell® Thin-Wall 384-Well Skirted PCR Plates: Bio-Rad; HSP3905

[0047] Microseal B Adhesive Sealer: Bio-Rad; MSB1001 Example

[0048] 1. Preparation of test samples: The DNA concentration of the test samples is required to be between 0-10 ng / μL. If the concentration is ≤ 10 ng / μL, 3.8 μL can be directly taken for subsequent reactions. If the concentration is > 10 ng / μL, the DNA needs to be diluted to 0-10 ng / μL with enzyme-free water, and 3.8 μL is taken for subsequent reactions.

[0049] 2. Probe Primer Design

[0050] By comparing the specific sequences in the genomes of Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, and Mycobacterium abscessus on the website of the National Center for Biotechnology Information (NCBI), we found highly conserved nucleotide sequences, and combined with the literature, we designed specific quadruple fluorescent PCR primers and probes for Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, and Mycobacterium abscessus. The designed primer and probe nucleotide sequences are shown in Table 1. Table 2 shows the probe and primer dosage of the Panel.

[0051] Table 1 Primer and probe information of four pathogenic microorganisms

[0052]

[0053] Table 2 Panel probe and primer amounts

[0054]

[0055] 3. Preparation of reference substances

[0056] Positive control: Select positive samples with known microbial species in clinical samples and whose microbial categories are consistent with those of the microorganisms to be tested. The concentration of the positive control samples before mixing is 0-10 ng / μL and enters the reaction system as positive control. Use the corresponding multiplex qPCR primers and probes for amplification. If there is an obvious S-type amplification curve, the consistency of the three replicate wells is good and the Ct value is <30, then the DNA mixture can continue to be used as a positive control for subsequent qPCR verification.

[0057] Negative control: Enzyme-free water is added to the reaction system instead of clinical samples as a negative control. Other reagents or reaction conditions are consistent with those of clinical samples.

[0058] 4. PCR amplification and signal collection

[0059] A 10 μL real-time fluorescence PCR amplification system was selected, and the reaction system is shown in Table 3.

[0060] Table 3 Multiplex qPCR reaction system

[0061]

[0062] PCR amplification conditions are:

[0063] Table 4 Multiplex qPCR reaction procedure

[0064]

[0065] 5. Interpretation of results

[0066] If the positive control and negative control meet the above conditions, the experiment is valid, otherwise the experiment needs to be repeated.

[0067] The sample to be tested has a Ct value and an obvious S-shaped amplification curve, indicating that the corresponding bacterial species of the sample is positively detected.

[0068] The sample to be tested has no Ct value and obvious S-shaped amplification curve, indicating that the corresponding bacterial species of the sample is negative.

[0069] Example 2 Comparison of the present invention with the single-plex fluorescent PCR method

[0070] The quadruple fluorescence PCR method of the present invention was compared with the single fluorescence PCR method using clinical positive samples. Figure 7 , Figure 8 It can be seen that there is no significant difference between the results of the quadruple fluorescence PCR system of the present invention and the single qPCR system.

[0071] Example 3 Evaluation of the specificity of the multiplex fluorescence PCR detection system

[0072] The multiplex fluorescence PCR method of the present invention was used to detect clinical samples that were negative for the four strains but contained other strains of the same genus. The results showed that except for the positive control, all other pathogens were negative.

[0073] Table 5 Clinical negative samples

[0074]

[0075] Example 4 Clinical sample testing

[0076] The multiplex fluorescence PCR method of the present invention was used to detect 10 clinical samples of infected patients, including alveolar lavage fluid, sputum and blood. The results are shown in Table 6, 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.

[0077] Table 6 Clinical sample test results

[0078]

[0079] The above-mentioned embodiments are only several preferred implementations of the present invention, and should not be construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the attached claims.

Claims

1. A primer probe set for multiplex qPCR detection of pathogens, It is characterized in that It includes a quadruple fluorescent PCR primer probe set suitable for the simultaneous detection of Aspergillus fumigatus, Staphylococcus aureus, Mycobacterium tuberculosis, and Mycobacterium abscessus in a single reaction; Primer set includes: A forward and reverse primer pair for detecting Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ ID NO.1-2; A forward and reverse primer pair for detecting Staphylococcus aureus, the nucleotide sequence of which is shown in SEQ ID NO.4-5; A forward and reverse primer pair for detecting Mycobacterium tuberculosis, the nucleotide sequence of which is shown in SEQ ID NO.7-8; A forward and reverse primer pair for detecting Mycobacterium abscessus, the nucleotide sequences of which are shown in SEQ ID NOs. 10-11; Probe sets include: 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 Staphylococcus aureus, the nucleotide sequence of which is shown in SEQ ID NO.6; A probe for reporting the amplified product of Mycobacterium tuberculosis, the nucleotide sequence of which is shown in SEQ ID NO.9; The probe used to report the amplified product of Mycobacterium abscessus, the nucleotide sequence of which is shown in SEQ ID NO.

12.

2. The primer probe set for multiplex qPCR detection of pathogens according to claim 1, It is characterized in that 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.

3. The primer probe set for multiplex qPCR detection of pathogens according to claim 1, It is characterized in that 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.

4. A kit, It is characterized in that It contains the primer probe set according to claim 1.

Images