A lamp primer composition for detecting pathogenic aspergillus and a kit thereof

Abstract

The application discloses a LAMP primer composition for detecting pathogenic aspergillus and a kit thereof, and the primer composition comprises external primers, internal primers and loop primers; and the sequences are specifically shown as SEQ ID NO:1-SEQ ID NO:6. The LAMP method provided by the application is shorter in time consumption than the traditional PCR method, does not need expensive and complex instruments, and is also applicable to primary hospitals. Compared with the culture method which takes 5-7 days to obtain results, the application can complete detection within 90 minutes, and more importantly, meets the strict requirement of detection time after instant acceptance of hospital environment disinfection (such as interval between operating rooms). The method of the application is high in specificity and sensitivity. The aspergillus LAMP primer composition is designed according to specific nucleic acid sequences of aspergillus, and interference of non-target bacteria and fungi is excluded, and the concentration of the least detectable genomic DNA is 10 fg / µL.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics, and more particularly to a LAMP primer composition and kit for detecting pathogenic Aspergillus. Background Technology

[0002] Invasive aspergillosis (IA) is a serious infection caused by fungi of the genus *Aspergillus*, with *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and *Aspergillus terreus* being the most common clinical causative agents. Due to its rapid progression, early and rapid diagnosis is crucial for improving patient prognosis. Furthermore, *Aspergillus* are widely present in hospital environments (such as in airborne dust, ventilation systems, and water supply pipes), and their spore aerosols are a major exogenous source of hospital-acquired infections in immunocompromised patients. Therefore, environmental hygiene monitoring in key areas such as laminar flow wards, intensive care units (ICUs), and operating rooms is a critical aspect of controlling IA.

[0003] However, current hospital environmental fungal monitoring mainly relies on standards such as the "Hospital Air Purification Management Standards," employing methods like plate settling or air sampler culture. These traditional methods are not only cumbersome to operate but also have long testing cycles, typically requiring 48 to 72 hours or even longer to observe colony growth and complete identification. This significant time lag means that monitoring results can often only serve as retrospective evaluations, failing to meet the clinical need for immediate early warning of environmental quality. Especially in clinical practice, after terminal disinfection of operating rooms or ICUs, medical staff urgently need to confirm whether the environment has met cleanliness standards in order to immediately admit the next patient. Existing culture methods cannot provide definitive results within a short timeframe (e.g., 1-2 hours), leading to blind spots in environmental safety assessments or wasting valuable medical resources while waiting for results.

[0004] Furthermore, although real-time quantitative PCR (qPCR) technology has been applied in clinical fungal detection with high sensitivity, its reliance on sophisticated thermal cycling instruments limits its widespread application in point-of-care testing (POCT) or on-site environmental monitoring. Meanwhile, some existing molecular diagnostic kits are often designed for only a single species (e.g., detecting only Aspergillus fumigatus), while there are many pathogenic Aspergillus species in the environment, including non-Aspergillus species such as Aspergillus flavus and Aspergillus terreus, which are also pathogenic. Using narrow-spectrum primers for environmental screening can easily lead to missed detections, creating a false impression of environmental safety.

[0005] In conclusion, there is an urgent need for a versatile, easy-to-use, and rapid-response detection technology in the fields of clinical diagnosis and hospital infection control. This technology should not only be applicable to the auxiliary diagnosis of clinical samples but also meet the specific needs of hospital environmental monitoring, particularly by enabling real-time detection before and after environmental disinfection. This would allow for the rapid evaluation of environmental cleanliness and provide strong technical support for preventing hospital-acquired aspergillosis. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a primer composition for the detection of pathogenic Aspergillus, its application, kit, and detection method, so as to achieve rapid and highly sensitive detection of Aspergillus species and immediate evaluation of environmental hygiene.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a LAMP primer composition for detecting pathogenic Aspergillus, the primer composition comprising an outer primer, an inner primer, and a loop primer; The external primers include the F3 primer with the sequence shown in SEQ ID NO:1 and the B3 primer with the sequence shown in SEQ ID NO:2; the internal primers include the FIP primer with the sequence shown in SEQ ID NO:3 and the BIP primer with the sequence shown in SEQ ID NO:4; the loop primers include the LF primer with the sequence shown in SEQ ID NO:5 and the LB primer with the sequence shown in SEQ ID NO:6.

[0008] Specifically, the primer composition specifically targets Aspergillus spp. ( Aspergillus The conserved region of the 28S rRNA gene of ribosomal DNA (spp.) contains the gene of *Pan-* Aspergillus Detection capability; the primer composition can detect at least the following Aspergillus species: Aspergillus fumigatus (… A . fumigatus Aspergillus flavus A . flavus Aspergillus niger (), A . niger Aspergillus terreus ( ) A . terreus Aspergillus nidus ( ) A . nidulans ), Aspergillus variegata ( A . versicolor ).

[0009] Specifically, the molar ratio of the outer primer, inner primer, and loop primer in the LAMP primer composition is 1:(5-9):(1-5); preferably 1:8:5.

[0010] The second aspect is to provide a kit for detecting pathogenic Aspergillus, comprising the LAMP primer composition described in the first aspect.

[0011] The kit also includes LAMP reaction reagents; the LAMP reaction reagents include Bst DNA polymerase, dNTPs, reaction buffer, and detection indicator; the detection indicator is selected from SYBR Green fluorescent dyes, calcein-manganese ion colorimetric solution, or hydroxynaphthol blue colorimetric solution.

[0012] Specifically, by integrating and optimizing the LAMP reaction, the Bst DNA polymerase (a complex enzyme preparation) and fluorescent dye used for the LAMP reaction can be in the form of lyophilized microspheres.

[0013] The third aspect is to provide the application of the LAMP primer composition described in the first aspect or the kit described in the second aspect in the preparation of pathogenic Aspergillus detection products.

[0014] Specifically, the detection product is used for the clinical auxiliary diagnosis of invasive aspergillosis; or, the detection product is used for monitoring hospital environmental hygiene and evaluating disinfection effectiveness to screen for Aspergillus contamination in the environment.

[0015] The fourth aspect is to provide a method for detecting pathogenic Aspergillus, which uses the kit described in the second aspect, and the detection method includes the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Perform isothermal amplification reaction using the kit; (3) Determine the detection results through fluorescence signals; The sample to be tested is a clinical biological sample or an environmental biological sample, and the detection method is a non-diagnostic method.

[0016] Specifically, the temperature of the isothermal amplification reaction is 60℃-70℃; the time of the isothermal amplification reaction is 20-60 minutes.

[0017] Specifically, in step (2), Bst The final concentration of DNA polymerase is 1-16 U, and the final concentration of dNTPs is 0.1-5 M.

[0018] Specifically, the clinical biological samples are selected from bronchoalveolar lavage fluid, sputum, blood, or tissue samples; the environmental biological samples are selected from air sampling filters, surface wiping fluid, or water samples.

[0019] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: The LAMP method provided by this invention is faster than the traditional PCR method (LAMP detection takes about 45 minutes, PCR detection takes about 120 minutes), and the LAMP reaction does not require expensive and complex instruments, making it more convenient and suitable for primary hospitals. Compared to the culture method which takes 5-7 days to produce results, this invention can complete the detection within 90 minutes. More importantly, it meets the stringent requirements of immediate acceptance after hospital environmental disinfection (such as the interval between consecutive operating rooms) regarding detection time.

[0020] The method of this invention has high specificity and sensitivity. The pan-Aspergillus LAMP primer composition is designed for the specific nucleic acid sequence of Aspergillus, eliminating interference from non-target bacteria and fungi, and can detect genomic DNA at a minimum concentration of 10 fg / μL (approximately 0.3-3 copies / μL).

[0021] The kit provided by this invention has been tested on a variety of clinical samples. The LAMP method provided is tolerant to common inhibitors in clinical samples and can be directly used on clinical samples such as bronchoalveolar lavage fluid, sputum and blood. Attached Figure Description

[0022] Figure 1 This figure shows the specificity verification results of the LAMP system of this invention with other non-target fungi (such as Candida and Mucor).

[0023] Figure 2 This is a sensitivity detection diagram of the primer composition of the present invention against Aspergillus fumigatus.

[0024] Figure 3 This is a sensitivity detection diagram of the primer composition of the present invention against Aspergillus flavus.

[0025] Figure 4 This is a sensitivity detection diagram of the primer composition of the present invention against Aspergillus niger.

[0026] Figure 5 This is a sensitivity detection diagram of the primer composition of the present invention against Aspergillus terrestris.

[0027] Figure 6 This is a sensitivity detection diagram of the primer composition of the present invention against Aspergillus nidulans.

[0028] Figure 7 This is a sensitivity detection diagram of the primer composition of the present invention against Aspergillus versicolor. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0030] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0031] Example 1 1. Primer design The conserved 28S rRNA regions of the genus *Aspergillus* were obtained from the database of the National Center for Biotechnology Information (NCBI). The rRNA genes were: *Aspergillus fumigatus* (GenBank ID: NG_055745.1); *Aspergillus flavus* (GenBank ID: NG_055742.1); *Aspergillus niger* (GenBank ID: NG_055744.1); *Aspergillus terreus* (GenBank ID: NG_055723.1); *Aspergillus nidus* (GenBank ID: NG_055739.1); *Aspergillus versicolor* (GenBank ID: NG_055743.1); *Aspergillus oryzae* (GenBank ID: AB105346.1); and *Aspergillus lanceolata* (GenBank ID: AB105346.1). Using the common sequences (GenBank ID: NG_070579.1; Aspergillus pyrolyticus GenBank ID: U29791.1; Aspergillus sylvaticus GenBank ID: AM883159.1; Aspergillus glaucus GenBank ID: NG_068980.1; Aspergillus gleamingus GenBank ID: LT626951.1; Aspergillus winterii GenBank ID: EF652157.1) as target sequences, LAMP primer compositions including external primer pairs, internal primer pairs, and loop primer pairs were designed.

[0032] The external primer pair includes external forward primer F3 and external reverse primer B3, the internal primer pair includes internal forward primer FIP and internal reverse primer BIP, and the loop primers include loop forward primer LF and loop reverse primer LB. The specific sequences are shown in Table 1 below.

[0033] Table 1

[0034] 2. Specificity test 1) Reagent preparation: Prepare a 10× primer mixture using the primers in Table 1 above. The primer ratios are shown in Table 2 below: Table 2

[0035] 2) The ratio of primers, SYBR Green I, and test samples in the LAMP reaction system is shown in Table 3 below: Table 3

[0036] The LAMP reaction lyophilized microspheres can be purchased LAMP Probe Lyophilized Beads (LBE323).

[0037] 3) The steps for detecting various Aspergillus species, other common pathogenic respiratory fungi, bacteria, and human genomic DNA using the above-mentioned Aspergillus pan-LAMP primer combination are as follows: ① Prepare the LAMP reaction system by mixing LAMP lyophilized microspheres, 10× primer mixture, SYBR GREEN I and the sample to be tested according to the ratio in Table 3 above; ② Place the prepared LAMP reaction system into a portable PCR instrument (commercially available), react at 63℃ for 45 min, and collect fluorescence every 1 min; ③ After the reaction is complete, interpret the results on the instrument.

[0038] Test results are as follows Figure 1 As shown, this method can be used to detect pathogenic Aspergillus.

[0039] Specifically, the standard strains include: *Aspergillus fumigatus* (CICC 41022), *Aspergillus flavus* (CICC 2436), *Aspergillus niger* (CICC 2485), *Aspergillus terreus* (ATCC 10690), *Aspergillus nidus* (CGMCC 3.3915), *Aspergillus versicolor* (CGMCC 3.3885), *Candida albicans* (ATCC 10231), *Candida glabrata* (ATCC 2001), *Candida parapsilosis* (ATCC 22019), *Candida tropicalis* (ATCC 750), *Candida krusei* (ATCC 6258), *Cryptococcus neoformans* (ATCC 14116), *Rhizopus umbellatus* (ATCC 44568), *Rhizopus spp.* (ATCC 22754), *Microsporum spp.* (ATCC 56018), *Klebsiella pneumoniae* (ATCC 13883), and *Acinetobacter baumannii* (ATCC 13883). The following bacteria were provided by the ATCC Biostandard Resource Center and the Shanghai Center for Microbiological Preservation: Pseudomonas aeruginosa (ATCC 27853), Haemophilus influenzae (ATCC 49247), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (ATCC 19615), Escherichia coli (ATCC 8739), and human mononuclear cells (THP-1).

[0040] Example 2 Sensitivity Test Based on Example 1 above, the genomic DNA of the six Aspergillus species provided in Example 1 was serially diluted 10-fold to: 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL. The different concentrations of genomic DNA were amplified using the LAMP reaction described in Example 1.

[0041] Test results are as follows Figure 2-7 As shown, all positive samples had a Tt value <40 min and exhibited typical amplification curves, thus they were interpreted as positive. Samples with concentrations below 100 fg / μL and negative controls (without DNA template) showed no amplification and were interpreted as negative. All sensitivity experiments were independently repeated three times. These results demonstrate that the method of this invention can detect pathogenic Aspergillus with high sensitivity.

[0042] Example 3 Based on Example 1 above, this example provides examples of detecting Aspergillus in clinical and environmental samples using the above-described method for detecting pathogenic Aspergillus. In the clinical sample testing, 63 samples were positive for Aspergillus culture and 39 samples were negative for Aspergillus culture, among which Aspergillus fumigatus was detected in 26 cases, Aspergillus flavus in 21 cases, Aspergillus niger in 13 cases, and Aspergillus terreus in 3 cases. In the environmental sample testing, 5 samples were positive for Aspergillus culture and 9 samples were negative for Aspergillus culture, among which Aspergillus flavus in 3 cases and Aspergillus niger in 3 cases.

[0043] 1. Clinical sample testing methods: Pretreatment: Sputum and bronchoalveolar lavage fluid samples were pretreated with a 4% (w / v) NaOH solution; whole blood samples were pretreated with erythrocyte lysis buffer.

[0044] Step 1: Extract genomic DNA using a commercial kit or automated nucleic acid extractor; The second step is to prepare a 10× primer mixture using the primers in Table 1 above. The primer combinations are shown in Table 2. Step 3: Place the prepared LAMP reaction system (as shown in Table 3) into a portable PCR instrument (commercially available), react at 63℃ for 45 min, and collect fluorescence every 1 min. Fourth step: After the reaction is complete, interpret the results on the instrument.

[0045] The test results are shown in Table 4 below, proving that the method of the present invention can complete the detection of pathogenic Aspergillus in sputum and bronchoalveolar lavage fluid samples.

[0046] Table 4

[0047] 2. Environmental sample testing methods: Collection and enrichment: Bioaerosols were collected using a liquid impact sampler (commercially available) and the sampled liquid was enriched using a centrifuge (8000 rpm, 5 min).

[0048] Step 1: Extract genomic DNA using a commercial kit or automated nucleic acid extractor; The second step is to prepare a 10× primer mixture using the primers in Table 1 above. The primer combinations are shown in Table 2. Step 3: Place the prepared LAMP reaction system (as shown in Table 3) into a portable PCR instrument (commercially available), react at 63℃ for 45 min, and collect fluorescence every 1 min. Fourth step: After the reaction is complete, interpret the results on the instrument.

[0049] The test results are shown in Table 5 below, proving that the method of the present invention can complete the detection of pathogenic Aspergillus in air aerosol samples.

[0050] Table 5

[0051] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A LAMP primer composition for detecting pathogenic Aspergillus, characterized in that, The primer composition includes external primers, internal primers, and loop primers; The external primers include the F3 primer with the sequence shown in SEQ ID NO:1 and the B3 primer with the sequence shown in SEQ ID NO:2; The inner primers include the FIP primer with the sequence shown in SEQ ID NO:3 and the BIP primer with the sequence shown in SEQ ID NO:4; the loop primers include the LF primer with the sequence shown in SEQ ID NO:5 and the LB primer with the sequence shown in SEQ ID NO:

6.

2. The LAMP primer composition for detecting pathogenic Aspergillus according to claim 1, characterized in that, The primer composition specifically targets the conserved region of the 28S rRNA gene of Aspergillus spp. ribosomal DNA, and has the ability to detect Pan-Aspergillus. The Aspergillus species that the primer composition can detect include at least: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, and Aspergillus versicolor.

3. The LAMP primer composition for detecting pathogenic Aspergillus according to claim 1, characterized in that, The molar ratio of the outer primer, inner primer, and loop primer in the LAMP primer composition is 1:(5-9):(1-5).

4. A kit for detecting pathogenic Aspergillus, characterized in that, Includes the LAMP primer composition according to any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, It also includes LAMP reaction reagents; the LAMP reaction reagents include Bst DNA polymerase, dNTPs, reaction buffer and detection indicator; the detection indicator is selected from SYBR Green fluorescent dyes, calcein-manganese ion colorimetric solution or hydroxynaphthol blue colorimetric solution.

6. The use of the LAMP primer composition according to any one of claims 1-3 or the kit according to any one of claims 4-5 in the preparation of pathogenic Aspergillus detection products.

7. The application according to claim 6, characterized in that, The detection product is used to provide in vitro detection information for clinical judgment; or, the detection product is used for monitoring hospital environmental hygiene and evaluating disinfection effectiveness to screen for Aspergillus contamination in the environment.

8. A method for detecting pathogenic Aspergillus, comprising using the kit described in claim 4 or 5, characterized in that, The detection method includes the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Perform isothermal amplification reaction using the kit; (3) Determine the detection results through fluorescence signals; The sample to be tested is a clinical biological sample or an environmental biological sample, and the detection method is a non-diagnostic method.

9. The detection method according to claim 8, characterized in that, The isothermal amplification reaction is carried out at a temperature of 60℃-70℃ and for a duration of 20-60 minutes.

10. The detection method according to claim 8, characterized in that, The clinical biological samples are selected from bronchoalveolar lavage fluid, sputum, blood, or tissue samples; the environmental biological samples are selected from air sampling filters, object surface wiping fluid, or water samples.

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