Narrowed food single cell detection universal primer pair and application thereof

Abstract

The present application relates to the technical field of nucleic acid detection, and discloses a general primer pair for detecting the genus of Segniliparus and application thereof. The detection primer pair and identification method provided by the present application are applicable to all species of the genus of Segniliparus, including but not limited to Segniliparus rugosus, and especially unnamed ANI species. The amplification method provided by the present application has low requirements for templates, and is applicable to clinical original samples such as sputum, alveolar lavage fluid, pleural and peritoneal fluid, culture, and environmental original samples such as water, soil and air, and extracted nucleic acid, and does not depend on culture. The method is fast and low-cost, and has low requirements for equipment and detection personnel, and is applicable to laboratories at all levels. The detection reagent based on the present application can be widely applied to related fields such as environmental monitoring, clinical precise detection and epidemiological research.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and more specifically, to universal primer pairs for detecting Stenotrophomonas spp. and their applications. Background Technology

[0002] genus Stenotrophomonas ( Stenotrophomonas (Also known as oligotrophomonas) belongs to the class Gamma-Proteobacteria, order Lysozymeles, family Lysozymeceae. A representative species is *Stenotrophomonas maltophilia*. S. maltophilia Gram-negative bacteria (GNP) have consistently been among the six most prevalent Gram-negative bacteria in clinical practice in my country, ranking 2nd-3rd among non-fermenting bacteria. They are consistently among the top 20 most common pathogens in various clinical samples, making them a major cause of nosocomial infections. Due to their multidrug resistance, high mortality rate, and exceptional biofilm-forming ability, [they are prone to infection]. S. maltophilia It is considered a marker of deteriorating lung function and a fatal disease for patients in intensive care, cancer, and those with weakened immune systems. The World Health Organization will... S. maltophilia It is classified as one of the most concerning emerging multidrug-resistant (MDR) Gram-negative bacteria globally. In addition to clinical testing, *Stenotrophomonas maltophilia* is a species monitored for public health and health hazard factors in multiple provinces of my country.

[0003] Stenotrophomonas bacteria also possess a variety of clearly defined biocontrol functions, including resistance to fungi, viruses, and beet armyworms; nitrogen fixation; promoting phosphate dissolution and plant growth; altering the composition of rhizosphere microorganisms; and degrading various chemical pesticides, insecticides, and environmental pollutants. Most species are considered emerging sources of plant rhizosphere growth promoters and biodegradable agents, as well as alternatives to synthetic fungicides. They are key microorganisms for observation, exploration, and utilization in environmental bioprotection and bioremediation.

[0004] Currently, based on the average nucleotide identity (ANI) of the whole genome, the genus *Stenotrophomonas* contains 116 species with ANIs, of which 38 are named species in the taxonomy (Frontiers in Microbiology, 2024, 12(5):1488674). Due to significant differences in phenotypes (including colony morphology, pigment and hemolysin production, growth temperature, etc.) and biochemical reactions between and within species, traditional phenotypic and biochemical identification methods are not very suitable for the genus *Stenotrophomonas*. The detection and identification of *Stenotrophomonas* employs molecular biology methods, mainly including: 1) 16S rRNA universal primer amplification and sequencing; 2) MLST typing of *Stenotrophomonas maltophilia* (based on...). atpD , gapA , guaA , mutM , new , ppsA , recA Sequence); 3) Other conserved genes, such as ssrA , chitA Detection methods for PCR, RT-PCR, and LAMP, etc.; 4) Mass spectrometry; 5) Whole genome sequencing.

[0005] The existing methods have the following problems:

[0006] First, *Stenotrophomonas* is a multispecies genus widely distributed in the natural environment. In clinical samples, among 116 ANI species (including 38 named species), only *Stenotrophomonas maltophilia* was found. S. maltophilia In addition to ), Stenotrophomonas septicemia ( S. sepilia ), Stenotrophomonas parvatinis ( S. pavanii ), Stenotrophomonas acidophilus ( S. acidophilus ), Stenotrophomonas rhizophila ( S. rhizophila Stenotrophomonas riyaki ( S. from Riyadh ), pigment-producing stenotrophozoites ( S. pigmentata ), Stenotrophomonas pleurisy S. tuberculopleurisy Multiple strains, including Stenotrophomonas maltophilia, have been reported to be isolated and cause pathogenicity. Furthermore, its biocontrol applications extend beyond Stenotrophomonas maltophilia. However, current detection / monitoring technologies for both single and multiple pathogens are primarily limited to Stenotrophomonas maltophilia.

[0007] Second, the average nucleic acid similarity analysis of 2213 *Stenotrophomonas* genomes in the NCBI database showed that 46% of the *Stenotrophomonas* genomes had incorrect species annotations, with *Stenotrophomonas maltophilia* accounting for the highest proportion (93%). 850 *Stenotrophomonas maltophilia* genomes were submitted after species correction. Furthermore, a key characteristic of *Stenotrophomonas* is the significant interspecies and intraspecies phenotypic and genomic variability. Therefore, primers designed and tested based on incorrectly annotated genomes, or primers designed without including the entire genome in the analysis, may be unsuitable for detecting *Stenotrophomonas*, and even *Stenotrophomonas maltophilia*.

[0008] Third, in addition to the two fundamental problems mentioned above, the main shortcomings of current methods / techniques for detecting Stenotrophomonas maltophilia are as follows: 1) 16S rRNA universal primer amplification and sequencing: only applicable to pure cultures, not suitable for direct detection of samples containing multiple microorganisms, such as environmental and clinical samples. 2) Including the MLST typing scheme for Stenotrophomonas maltophilia. atpD , gapA , guaA , mutM , new , ppsA , recA Genes (PubMLST, https: / / pubmlst.org / organisms / stenotrophomonas-maltophilia / primers) and others such as ssrA , chitA Detection methods for conserved genes using PCR, RT-PCR, and LAMP: Primers generally have high specificity, but insufficient sensitivity, and cannot detect all Stenotrophomonas species, or even all Stenotrophomonas maltophilia. 3) Mass spectrometry: Currently the most commonly used method in clinical practice, but the built-in comparison database only contains... S. acidaminiphila , S. maltophilia , S. rhizophila , S. nitrite reductans and S. of the painters Five bacterial species were tested, but the false negative rate was high, and the error rate in species identification was even higher. 4) Whole genome sequencing: the most accurate method for species identification, but not suitable for rapid testing or direct detection of mixed environmental and clinical samples.

[0009] Therefore, there is an urgent need to establish a rapid detection method that can cover all species of the Stenotrophomonas genus for the detection and monitoring of environmental and clinical samples, thereby improving the detection rate. Summary of the Invention

[0010] The purpose of this invention is to provide universal primer pairs for the detection of Stenotrophomonas spp. and their applications.

[0011] To achieve the objectives of this invention, in a first aspect, this invention provides *Stereotrophozoa* spp. ( Stenotrophomonas ) Detection of universal primer pairs, including (SEQ ID NO:1-2):

[0012] Upstream primer: 5'-ATCCAYAVCGACAAGATCCT-3';

[0013] Downstream primer: 5'-ACGTTGTGRTGRCTCTTGA-3';

[0014] In this context, Y represents the base C or T, V represents the base A, C or G, and R represents the base G or A.

[0015] Secondly, the present invention provides detection reagents or kits containing the primer pairs.

[0016] Thirdly, the present invention provides a kit for detecting Stenotrophomonas spp., comprising the primer pair, and further comprising at least one of DNA polymerase, dNTPs and PCR reaction buffer.

[0017] Fourthly, the present invention provides the use of the primer pair or the detection reagent or kit in the preparation of products for detecting Stenotrophomonas spp. in samples.

[0018] Fifthly, the present invention provides a method for detecting the presence of Stenotrophomonas bacteria in a sample (including non-disease diagnosis and treatment purposes), comprising the following steps:

[0019] (1) Extract nucleic acid from the sample to be tested;

[0020] (2) Using the extracted nucleic acid as a DNA template, a nucleic acid amplification reaction is carried out using the primer pair;

[0021] (3) Detect whether nucleic acid amplification products are generated in step (2). If nucleic acid amplification products are detected, it indicates that there are Stenotrophomonas bacteria in the sample.

[0022] Furthermore, the nucleic acid amplification reaction is a polymerase chain reaction (PCR).

[0023] Furthermore, the size of the nucleic acid amplification product is approximately 1061 bp.

[0024] Furthermore, the sample can be a clinical sample or an environmental sample.

[0025] The clinical sample can be selected from one of the following: sputum, bronchoalveolar lavage fluid, pleural and peritoneal fluid, and bacterial cultures.

[0026] The environmental sample can be selected from one of the following: water sample, soil sample, and air sample.

[0027] The *Stemonae* bacteria include, but are not limited to, the following named species: *Stemonae acidophilus* (… S. acidophilus ), Stenotrophomonas africanus ( S. africana ), Stenotrophomonas araceae ( S. aracearum ), Stenotrophomonas saponinosa ( S. bentonite ), Stenotrophomonas cirrhosa ( S. betel ), Stenotrophomonas capsici ( S. capsicum ), chelates of Stenotrophomonas ( S. chelatiphaga ), Stenotrophomonas quercetin ( S. cyclobalanopsis ), Stenotrophomonas daetianense ( S. daejeonensis ), S. detusculanense Stenotrophomonas forensic medicine ( S. forensis ), Stenotrophomonas hygroscopicus ( S. frigidarius ), Stenotrophomonas geniculata ( S. kneeling ), Stenotrophomonas ginseng ( S. ginsengisoli ), Stenotrophomonas hibiscus (S. hibiscus ), Stenotrophomonas spore-forming bacteria ( S. humi ), indicator stenotrophomonas ( S. indicatrix ), S. indolent Korean Stenotrophomonas ( S. koreensis ), Stenotrophomonas lactifera ( S. lactitubi ), Stenotrophomonas erhaiensis ( S. lacuserhaii Stenotrophomonas maltophilia ( S. maltophilia ), Stenotrophomonas genus ( S. to die Stenotrophomonas muscularis ( S. muris ), Stenotrophomonas nematodes ( S. nematodicola ), reduced nitrite-sparing stenotrophomonas ( S. nitrite reductans ), Oahu Stenotrophomonas ( S. oahuensis ), Stenotrophomonas ginseng ( S. panacihumi ), Stenotrophomonas parvatinis ( S. pavanii ), Stenotrophomonas picketii ( S. pictorum ), pigment-producing stenotrophozoites ( S. pigmentata ), Stenotrophomonas rhizophila ( S. rhizophila Stenotrophomonas riyaki ( S. riyadhensis ), Stenotrophomonas septicemia ( S. sepilia ), Stenotrophomonas strophinae ( Holy land ), Stenotrophomonas thermophilus ( S. thermophila ), Stenotrophomonas pleurisy S. tuberculopleuritidis ), Stenotrophomonas gracilis ( S. tumulicola ).

[0028] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0029] (i) It has high sensitivity and can detect all Stenotrophomonas in the sample, indicating the clinical pathogenic risk of Stenotrophomonas, not limited to Stenotrophomonas maltophilia, to patients. At the same time, it more accurately reflects the diversity of Stenotrophomonas species in environmental samples.

[0030] (ii) This invention is based on PCR technology for detection, and the sensitivity of the PCR method itself is 1×10⁻⁶. 5 CFU / mL, increased to 1×10 after enrichment. 0 CFU / mL. It is also low-cost, fast, with a detection time of 0.5-1.5 hours, simple to operate, and has low requirements for equipment and testing personnel, making it more widely applicable in laboratories at all levels.

[0031] (iii) The amplification method has low requirements for templates and can be used for original samples such as sputum, bronchoalveolar lavage fluid, pleural effusion, ascites, and cultures, as well as extracted nucleic acids. It does not depend on culture and can shorten the clinical diagnosis time. Attached Figure Description

[0032] Figure 1 The above is an electrophoresis image of PCR amplification of 20 Stenotrophomonas strains in a preferred embodiment of the present invention; wherein, M: DNA molecular weight marker; N: physiological saline negative control; the information of the strains corresponding to lanes 1 to 20 is shown in Table 1. Detailed Implementation

[0033] The present invention aims to provide a rapid and highly sensitive primer pair and identification method covering all species of the Stenotrophomonas genus, to improve the detection rate of Stenotrophomonas in environmental and clinical sample detection and monitoring, and to avoid missed diagnoses / detections.

[0034] The present invention adopts the following technical solution:

[0035] The genomes were annotated using Prokka (with the `-genus` and `--species` parameters specifying the genus and species). The output GFF file was used as input for Roary, and pan-genome analysis and multiple sequence alignment of core genes were performed on reference genomes of 116 *Streptomyces* species, yielding 11 core genes. guaA The gene is also used in PubMLST for Stenotrophomonas maltophilia ( S. maltophilia One of the seven genes used in MLST typing. (Vasileuskaya-Schulz Z, Kaiser S, Maier T, Kostrzewa M, Jonas D. Delineation of Stenotrophomonas spp. by multi-locus sequence analysis and MALDI-TOF mass spectrometry. Syst Appl Microbiol. 2011;34(1):35-39. doi:10.1016 / j.syapm.2010.11.011), was used as the preferred gene for primer design.

[0036] Stenotrophomonas maltophilia NCTC 10257 (GCF_900186865.1) guaAThe gene, used as a reference sequence, is located at NZ_LT906480.1:2204368-2205933 (+), and is numbered CKW06_RS10705 in Refseq annotation version GCF_900186865.1-RS_2025_05_06, with a full length of 1568 bp. The genomes of 116 *Streptomyces* ANI strains were aligned and extracted using BLAST v2.16.0+. guaA Genes. Alignment was performed using Muscle v5.3 to identify Stenotrophomonas genus. guaA By targeting conserved regions of the gene, specific primer pairs were designed to achieve specific amplification of *Stenophora* spp. while avoiding non-specific amplification products from other genera. After comprehensive comparison, primers located in... guaA The sequence at 10-29 (+) of the gene, with the mutation point at 5'-3', is: ATC CA(C / T) A(A / C / G)C GAC AAG ATC CT; downstream at 1052-1070 (-), the sequence at 5'-3' is: ACG TTG TG(G / A) T(G / A)G CTC TTG A. The sequences after degenerate base pairing are: ST-guaA YW-F: ATC CAY AVC GAC AAG ATC CT; ST-guaA YW-L: ACG TTG TGR TGRCTC TTG A, with a product size of approximately 1061 bp.

[0037] Clone Manager software was used to detect primer binding on all reference genomes and NCBI-submitted genomes, as well as simulated PCR amplification. The online Primer Blast Core nucleotide database (core_nt) was used for searching, without limiting the organism or the family Lysozyme. Lysobacteraceae Annealing temperatures were set to Min 58℃ and Max 63℃ to test primer specificity. Results showed that the ST-guaA YW-F / L primer pair could amplify all species of the *Streptomyces* genus; the upstream and downstream primers were effective against *Xanthomonas* species within the same family (…). Xanthomonas ), Xanthomonas spp. Pseudoxanthomonas ), Lysozyme ( Lysobacterium Some strains have binding on their genome, but the product sequence can be completely distinguished from the genus Stenotrophomonas, exhibiting high specificity.

[0038] This invention provides a universal primer pair and identification method for detecting Stenotrophomonas spp., the primers being as follows (SEQ ID NO: 1-2):

[0039] ST-guaA YW-F:5′-ATCCAYAVCGACAAGATCCT-3′

[0040] ST-guaA YW-R: 5′-ACGTTGTGRTGRCTCTTGA-3′

[0041] In this context, Y represents the base C or T, V represents the base A, C or G, and R represents the base G or A.

[0042] The detection primer pairs and identification methods provided by this invention are applicable to all species of the genus Stenotrophomonas, including but not limited to Stenotrophomonas maltophilia, especially unnamed ANI species.

[0043] The amplification method provided by this invention has low requirements for templates and is applicable to clinical raw samples such as sputum, bronchoalveolar lavage fluid, pleural effusion, ascites, and cultures, as well as environmental raw samples such as water, soil, and air, and the extracted nucleic acids. It does not depend on culture.

[0044] This method is fast and low-cost, with low requirements for equipment and testing personnel, and can be applied to laboratories at all levels.

[0045] The detection reagents based on this invention can be widely used in environmental monitoring, clinical precision testing, and epidemiological research and other related fields.

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] Example 1: Detection of actual bacterial cultures

[0048] 1) Twenty strains of Stenotrophomonas strains preserved by our research group (strain information is shown in Table 1) were inoculated into Luria-Bertani (LB) medium and cultured for 2 days. Fresh cultures were picked and added to 200 μL of physiological saline to prepare templates.

[0049] 2) Prepare a 20 μL reaction system using 2×EasyTaq® PCR SuperMix (TransGen Biotech). The components are as follows:

[0050]

[0051] 3) Amplification on the instrument, reaction conditions: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min) 30 cycles, 72℃ for 5 min, 4℃∞. Total time 70 min.

[0052] 4) 1% agarose gel electrophoresis, load 3 μL of sample, and read the results using a Bio-rad gel reader.

[0053] 5) All 20 bacterial strains amplified positively (see results below). Figure 1 ).

[0054] Table 1. 20 Stenotrophomonas species

[0055]

[0056] Note: — indicates an unnamed ANI strain.

[0057] Example 2: Environmental Sample Detection

[0058] 1) Collected 45 air samples, 8 air conditioning cooling water samples, and 100 domestic water samples (drinking water, hot water for showers, etc.), for a total of 153 samples, numbered HJ-1 to HJ-153.

[0059] 2) Centrifuge at 10000×g for 5 minutes, discard the supernatant, and add 20 mL of LB liquid medium for enrichment culture.

[0060] 3) Prepare the following 20μL reaction system using TSINGKE TSE101 Gold Mix (green) (Beijing Qingke Biotechnology Co., Ltd.):

[0061]

[0062] 4) Amplification on the instrument, reaction conditions: 98℃ for 2 min, (98℃ for 10 s, 58℃ for 10 s, 72℃ for 20 s) for 30 cycles, 72℃ for 5 min, 4℃ at ∞. Total time: 27 min.

[0063] 5) 1% agarose gel electrophoresis, load 3 μL of sample, and read the results using a Bio-rad gel reader.

[0064] 6) Simultaneously, streak the enrichment broth along the triple phase line into LB medium and isolate the colonies.

[0065] 7) Results: Samples HJ-28, HJ-40, and HJ-64 showed positive amplification. *Streptotrophozoites* were also isolated from these three samples on the streak plating medium, and two strains were identified as *Streptotrophozoites geniculateus*. S. geniculata One strain was *Stereotrophozoa hygroscopica* (Syntrophozoa hygroscopica). S. frigidarius Both sensitivity and specificity are 100%.

[0066] Example 3: Development and design of universal primer pairs for the detection of Stenotrophomonas spp.

[0067] exist guaA Three sets of primers were designed on the gene sequence:

[0068] Group 1 (SEQ ID NO:1-2): Product size is approximately 1061bp

[0069] ST-guaA (1)-F: 5′-ATCCAYAVCGACAAGATCCT-3′ (10-29bp)

[0070] ST-guaA (1)-R: 5′-ACGTTGTGRTGRCTCTTGA-3′ (1052-1070bp)

[0071] Group 2 (SEQ ID NO:3-4): Product size approximately 582bp

[0072] ST-guaA (2)-F: 5′-CATGGCCAACGAAGANAAGC-3′ (489-508bp)

[0073] ST-guaA (2)-R: 5′-ACGTTGTGRTGRCTCTTGA-3′ (1052-1070bp)

[0074] Group 3 (SEQ ID NO:5-6): Product size approximately 920bp

[0075] ST-guaA (3)-F: 5′-CATGGCCAACGAAGANAAGC-3′ (489-508bp)

[0076] ST-guaA (3)-R: 5′-CCCTTAGCGCAATNACCCA-3′ (1390-1408bp)

[0077] Where Y represents the base C or T, V represents the base A, C or G, R represents the base G or A, and N represents the base A, T, C or G.

[0078] Simulated amplification showed that, except for the first set of primers (i.e., the primers of this patent) which can amplify all species of the genus *Stenophobicomonas*, the second set of primers, limited by the upstream primer, can only amplify 95 species, and the third set of primers, limited by the downstream primer, can only amplify 59 species. Therefore, the first set of primers was selected in this invention.

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. *Stenophobic Monotrophus* genus ( Stenotrophomonas ) Detection of universal primer pairs, characterized in that, include: Upstream primer: 5'-ATCCAYAVCGACAAGATCCT-3'; Downstream primer: 5'-ACGTTGTGRTGRCTCTTGA-3'; In this context, Y represents the base C or T, V represents the base A, C or G, and R represents the base G or A.

2. A detection reagent or kit containing the primer pair of claim 1.

3. A kit for detecting Stenotrophomonas spp., characterized in that, It comprises the primer pair of claim 1, and further comprises at least one of DNA polymerase, dNTPs and PCR reaction buffer.

4. The use of the primer pair of claim 1 or the detection reagent or kit of claim 2 or 3 in the preparation of products for detecting Stenotrophomonas spp. in samples.

5. A method for detecting the presence of Stenotrophomonas bacteria in a sample, characterized in that, Includes the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Using the extracted nucleic acid as a DNA template, a nucleic acid amplification reaction is carried out using the primer pair described in claim 1; (3) Detect whether a nucleic acid amplification product of size 1061bp is generated in step (2). If a nucleic acid amplification product is detected, it indicates that there are Stenotrophomonas bacteria in the sample. The method described is not for disease diagnosis and treatment purposes.

6. The method according to claim 5, characterized in that, The nucleic acid amplification reaction is a polymerase chain reaction.

7. The method according to claim 5, characterized in that, The sample is an environmental sample.

8. The method according to claim 7, characterized in that, The environmental sample is selected from one of the following: water sample, soil sample, and air sample.

9. The method according to any one of claims 5-8, characterized in that, The *Streptococcus* genus bacteria includes the following named species: *Streptococcus acidophilus* (… S. acidaminiphila ), Stenotrophomonas africanus ( S. africana ), Stenotrophomonas araceae ( S. aracearum ), Stenotrophomonas saponinosa ( S. bentonitica ), Stenotrophomonas cirrhosa ( S. beteli ), Stenotrophomonas capsici ( S. capsici ), chelates of Stenotrophomonas ( S. chelatiphaga ), Stenotrophomonas quercetin ( S. cyclobalanopsidis ), Stenotrophomonas daetianense ( S. daejeonensis ), S. detusculanense Stenotrophomonas forensic medicine ( S. forensis ), Stenotrophomonas hygroscopicus ( S. frigidaeris ), Stenotrophomonas geniculata ( S. geniculata ), Stenotrophomonas ginseng ( S. ginsengisoli ), Stenotrophomonas hibiscus ( S. hibiscicola ), Stenotrophomonas spore-forming bacteria ( S. humi ), indicator stenotrophomonas ( S. indicatrix ), S. indologenes Korean Stenotrophomonas ( S. koreensis ), Stenotrophomonas lactifera ( S. lactitubi ), Stenotrophomonas erhaiensis ( S. lacuserhaii Stenotrophomonas maltophilia ( S. maltophilia ), Stenotrophomonas genus ( S. mori Stenotrophomonas muscularis ( S. muris ), Stenotrophomonas nematodes ( S. nematodicola ), reduced nitrite-sparing stenotrophomonas ( S. nitritireducens ), Oahu Stenotrophomonas ( S. oahuensis ), Stenotrophomonas ginseng ( S. panacihumi ), Stenotrophomonas parvatinis ( S. pavanii ), Stenotrophomonas picketii ( S. pictorum ), pigment-producing stenotrophozoites ( S. pigmentata ), Stenotrophomonas rhizophila ( S. rhizophila Stenotrophomonas riyaki ( S. riyadhensis ), Stenotrophomonas septicemia ( S. sepilia ), Stenotrophomonas strophinae ( S. terrae ), Stenotrophomonas thermophilus ( S. thermophila ), Stenotrophomonas pleurisy S. tuberculopleuritidis ), Stenotrophomonas gracilis ( S. tumulicola ).

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