Composition for detecting and typing salmonella paratyphi

Abstract

The invention belongs to the field of molecular biological detection, particularly relates to detection of salmonella paratyphi, and more particularly relates to detection of salmonella paratyphi A, salmonella paratyphi B and salmonella paratyphi C. The composition provided by the invention can specifically detect salmonella paratyphi only by detecting one target spot, the operation is simple, non-specific amplification does not exist, and particularly, salmonella paratyphi can be accurately distinguished from some genetically similar bacteria genus. And the sensitivity reaches 500 copies / mL, the detection is more accurate, a relatively sufficient basis for rapidly detecting and excluding infection of different pathogens can be provided, the detection time of the salmonella paratyphi is shortened, and the implementation of countermeasures is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of molecular biological detection, and in particular, relates to the detection of Salmonella paratyphi, and more particularly, to the detection of Salmonella paratyphi type A, B and C. Background Art

[0002] Enteric Salmonella serotypes typhoid and paratyphoid are restricted Gram-negative bacterial pathogens in humans that can cause severe blood infections, collectively known as enteric fever. Epidemics are mainly caused by contaminated water and food. Poor hygiene conditions and living habits are the main causes of the disease. However, with the increase in human communication, the incidence of enteric fever has gradually become a prominent health problem in most countries. Paratyphoid fever includes paratyphoid A, paratyphoid B and paratyphoid C. The pathogens of paratyphoid A, paratyphoid B and paratyphoid C belong to the A, B and C groups of Salmonella, respectively. Their biochemical characteristics are similar to those of Salmonella typhi, but the components of bacterial antigens and flagellar antigens are different. Although the pathogenesis and epidemic characteristics of paratyphoid and typhoid are generally similar, from the perspective of the widespread prevalence of paratyphoid in recent years, the clinical manifestations are often atypical and diverse, which makes it difficult to detect and control the disease in an early stage, thus easily causing the spread and prevalence of the epidemic. Therefore, early detection, early reporting, early isolation and early treatment are the key to controlling the epidemic.

[0003] The identification of Salmonella paratyphi is mainly based on biochemical tests and serotyping, and it takes 5 to 7 days to get the test results. However, the current public PCR method tools are limited in the application of routine monitoring of serotype Paratyphi. Some methods use multiple targets for joint judgment to distinguish, and some methods use single target detection but have low detection sensitivity or insufficient specificity. In addition, some studies have explored the application of enteric fever monitoring caused by Salmonella based on whole genome sequencing (WGS) genotyping (such as SISTR2, SeqSero2, Genotyphi, Paratype). However, due to the cost and the need for professional instruments and bioinformatics resources, the routine use of WGS in daily typing is greatly limited and requires further research and verification. With the development of molecular biotechnology, there have been continuous attempts to use invA, prt, fliC, tyV, flagellar antigen Hd, Vi antigen gene, STY1633 and SPA4829 as target genes, and to detect Salmonella Paratyphi using conventional PCR and fluorescent PCR. However, the above target genes are not specific genes of Salmonella Paratyphi. Some require the amplification combination of 2 to 3 genes for identification, and the operation is cumbersome and complicated; some are non-specific or have low coverage. For example, patent CN102102124A and Xu Yi's published article (DOI: 10.19813 / j.cnki.weishengyanjiu.2017.02.024), in which the specific amplification primer probe for detecting Salmonella paratyphi A not only matches the target species, but also completely matches other types such as FDAARGOS_688 (NZ_CP046280.1) and enterica serovarBispebjerg (NZ_CP115551.1). Patents CN102634577A and CN102102124A cannot distinguish the target from Salmonella enterica subsp. enterica serovar Bareilly. The patent CN102102124A related to Salmonella paratyphi B has been disclosed. The adjacent amplification segments selected in the article published by Xu have the situation that in addition to the target species, multiple types at the same classification level such as Typhimurium, Johannesburg, Minnesota, etc. are non-specifically amplified. The primer probe selected by Xu's published article for detecting Salmonella paratyphi C has the problem of non-specific amplification of Choleraesuis. Ma's research article (1672-3619 (2013) 07-0820-04) requires the comprehensive identification of multiple targets for Salmonella paratyphi, which is complicated.

[0004] Therefore, there is a need in the art for a product that only needs to detect one target, is simple to operate, and can specifically detect Salmonella paratyphi. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a composition for detecting and typing Salmonella paratyphi, comprising at least one of the following nucleic acid compositions:

[0006] A first nucleic acid composition comprising:

[0007] 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.1, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.2, and a probe arranged across the junction region of SEQ ID NO.1 and SEQ ID NO.2; or,

[0008] 2) an upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO.1 and SEQ ID NO.2;

[0009] A second nucleic acid composition comprising:

[0010] 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.3, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.4, and a probe arranged across the junction region of SEQ ID NO.3 and SEQ ID NO.4; or,

[0011] 2) an upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO. 3 and SEQ ID NO. 4; or

[0012] A third nucleic acid composition comprises:

[0013] 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.5, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.6, and a probe arranged across the junction region of SEQ ID NO.5 and SEQ ID NO.6; or,

[0014] 2) An upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO. 5 and SEQ ID NO. 6.

[0015] Furthermore, the composition of the present invention comprises at least two of any of the above-mentioned nucleic acid compositions.

[0016] Furthermore, the composition of the present invention comprises:

[0017] A first nucleic acid composition comprising:

[0018] 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.1, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.2, and a probe arranged across the junction region of SEQ ID NO.1 and SEQ ID NO.2; or,

[0019] 2) an upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO.1 and SEQ ID NO.2;

[0020] A second nucleic acid composition comprising:

[0021] 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.3, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.4, and a probe arranged across the junction region of SEQ ID NO.3 and SEQ ID NO.4; or,

[0022] 2) an upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO. 3 and SEQ ID NO. 4; and

[0023] A third nucleic acid composition comprises:

[0024] 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.5, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.6, and a probe arranged across the junction region of SEQ ID NO.5 and SEQ ID NO.6; or,

[0025] 2) An upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO. 5 and SEQ ID NO. 6.

[0026] A probe set across the connecting region of SEQ ID NO.1 and 2, SEQ ID NO.3 and 4, or SEQ ID NO.5 and 6 means that the sequence targeted by the probe is a continuous sequence that includes a portion of the sequences shown in SEQ ID NO.1 and 2, a continuous sequence that includes a portion of the sequences shown in SEQ ID NO.3 and 4, or a continuous sequence that includes a portion of the sequences shown in SEQ ID NO.5 and 6.

[0027] An upstream primer or downstream primer targeting the connecting region spanning SEQ ID NO.1 and 2, SEQ ID NO.3 and 4, or SEQ ID NO.5 and 6 means that the sequence targeted by the upstream primer or downstream primer includes a continuous sequence of a portion of the sequences shown in SEQ ID NO.1 and 2, a continuous sequence of a portion of the sequences shown in SEQ ID NO.3 and 4, or a continuous sequence of a portion of the sequences shown in SEQ ID NO.5 and 6.

[0028] Furthermore, the upstream primer in the first nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 7-9.

[0029] Furthermore, the downstream primer in the first nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 10-12.

[0030] Furthermore, the upstream primer in the second nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 13-15.

[0031] Furthermore, the downstream primer in the second nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 16-18.

[0032] Furthermore, the upstream primer in the third nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 19-21.

[0033] Furthermore, the downstream primer in the third nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 22-24.

[0034] Furthermore, the probe of the first nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 25-27.

[0035] Furthermore, the probe of the second nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 28-30.

[0036] Furthermore, the probe of the third nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 31-33.

[0037] In some specific embodiments, the first nucleic acid composition comprises at least one of the following combinations:

[0038] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.7, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.10, and a probe having a nucleotide sequence as shown in SEQ ID NO.25;

[0039] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.8, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.11, and a probe having a nucleotide sequence as shown in SEQ ID NO.26; or

[0040] The nucleotide sequence of the upstream primer is shown as SEQ ID NO.9, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.12, and the nucleotide sequence of the probe is shown as SEQ ID NO.27.

[0041] In some specific embodiments, the second nucleic acid composition comprises at least one of the following combinations:

[0042] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.13, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.16, and a probe having a nucleotide sequence as shown in SEQ ID NO.28;

[0043] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.14, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.17, and a probe having a nucleotide sequence as shown in SEQ ID NO.29; or

[0044] The nucleotide sequence of the upstream primer is shown as SEQ ID NO.15, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.18, and the nucleotide sequence of the probe is shown as SEQ ID NO.30.

[0045] In some specific embodiments, the third nucleic acid composition comprises at least one of the following combinations:

[0046] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.19, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.22, and a probe having a nucleotide sequence as shown in SEQ ID NO.31;

[0047] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.20, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.23, and a probe having a nucleotide sequence as shown in SEQ ID NO.32; or

[0048] The nucleotide sequence of the upstream primer is shown as SEQ ID NO.21, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.24, and the nucleotide sequence of the probe is shown as SEQ ID NO.33.

[0049] In a specific embodiment, the composition comprises:

[0050] A first nucleic acid composition comprising:

[0051] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.7, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.10, and a probe having a nucleotide sequence as shown in SEQ ID NO.25;

[0052] A second nucleic acid composition comprising:

[0053] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.13, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.16, and a probe having a nucleotide sequence as shown in SEQ ID NO.28;

[0054] A third nucleic acid composition comprises:

[0055] The nucleotide sequence of the upstream primer is shown as SEQ ID NO.19, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.22, and the nucleotide sequence of the probe is shown as SEQ ID NO.31.

[0056] In a specific embodiment, the composition comprises:

[0057] A first nucleic acid composition comprising:

[0058] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.8, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.11, and a probe having a nucleotide sequence as shown in SEQ ID NO.26;

[0059] A second nucleic acid composition comprising:

[0060] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.14, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.17, and a probe having a nucleotide sequence as shown in SEQ ID NO.29;

[0061] A third nucleic acid composition comprises:

[0062] The nucleotide sequence of the upstream primer is shown as SEQ ID NO.20, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.23, and the nucleotide sequence of the probe is shown as SEQ ID NO.32.

[0063] In a specific embodiment, the composition comprises:

[0064] A first nucleic acid composition comprising:

[0065] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.9, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.12, and a probe having a nucleotide sequence as shown in SEQ ID NO.27;

[0066] A second nucleic acid composition comprising:

[0067] An upstream primer having a nucleotide sequence as shown in SEQ ID NO.15, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.18, and a probe having a nucleotide sequence as shown in SEQ ID NO.30;

[0068] A third nucleic acid composition comprises:

[0069] The nucleotide sequence of the upstream primer is shown as SEQ ID NO.21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.24, and the nucleotide sequence of the probe is shown as SEQ ID NO.33.

[0070] The composition provided by the present invention can specifically detect Salmonella paratyphi by only detecting one target point, is simple to operate, does not have non-specific amplification, and can be used to distinguish Salmonella paratyphi from some genetically similar bacteria. In addition, the sensitivity reaches 500 copies / mL, the detection is more accurate, and can provide a relatively sufficient basis for rapid detection and exclusion of different pathogens, shorten the detection time of Salmonella paratyphi, and accelerate the implementation of countermeasures.

[0071] The present invention creatively discovered a very specific targeting sequence in Salmonella paratyphi type A, B, and C, and the targeting sequence includes a front part of a specific segment and a rear part of a specific segment. The front part and the rear part of the specific segment are directly connected, and the connecting part of the two is a connecting region, which is a sequence that is specifically conserved for the target species (except for the corresponding target type to be detected, the remaining types of Salmonella have more or less insertion sequences between the front part of the specific segment and the rear part of the specific segment, so primers and probes are designed according to this deletion, that is, the corresponding Salmonella paratyphi type A, B, and C can be accurately detected).

[0072] The method for detecting the junction region of the target sequence includes designing primers or probes for the junction region: 1) targeting the upstream primer or downstream primer across the junction region, when the detection object is not Paratyphoid Salmonella A, B, or C, the primer designed across the junction region cannot achieve amplification; or 2) designing an upstream primer targeting the front part of the specific segment, designing a downstream primer targeting the rear part of the specific segment, and setting a probe across the junction region; the amplification products of the upstream and downstream primers span the junction region, when the detection object is not Paratyphoid Salmonella A, B, or C, the probe designed across the junction region cannot mark the amplification product.

[0073] The term "joining region" refers to the sequence between the front and rear parts of the specific segment. In the corresponding target type strain, it refers to the direct connection between the front and rear parts of the specific segment, and the joining region has no nucleotide sequence, while in the other non-target Salmonella strains, the joining region has a certain nucleotide sequence.

[0074] Specifically, the specific targeting sequence of Salmonella paratyphi A is a nucleotide sequence as shown in SEQ ID NO.34, including the front part of the specific segment as shown in SEQ ID NO.1 and the rear part of the specific segment as shown in SEQ ID NO.2. In Salmonella paratyphi A, SEQ ID NO.1 and 2 directly constitute SEQ ID NO.34, and there is no nucleotide sequence in the connecting region, while in Salmonella paratyphi B and C, there are certain nucleotides between SEQ ID NO.1 and 2 as the connecting region. Designing amplification primers or probes across the connecting region of SEQ ID NO.1 and SEQ ID NO.2 can specifically detect Salmonella paratyphi A. For example, the upstream primer targets the nucleotide sequence shown in SEQ ID NO.1, the downstream primer targets the nucleotide sequence shown in SEQ ID NO.2, and the probe is set to span the connecting region of SEQ ID NO.1 and SEQ ID NO.2; or, the upstream primer or the downstream primer targets the connecting region across SEQ ID NO.1 and SEQ ID NO.2.

[0075] Specifically, the specific targeting sequence of Salmonella paratyphi B is a nucleotide sequence as shown in SEQ ID NO.35, including the front part of the specific segment as shown in SEQ ID NO.3 and the rear part of the specific segment as shown in SEQ ID NO.4. In Salmonella paratyphi B, SEQ ID NO.3 and 4 directly constitute SEQ ID NO.35, and there is no nucleotide sequence in the connecting region, while in Salmonella paratyphi A and C, there are certain nucleotides between SEQ ID NO.3 and 4 as the connecting region. Designing amplification primers or probes across the connecting region of SEQ ID NO.3 and SEQ ID NO.4 can specifically detect Salmonella paratyphi B. For example, the upstream primer targets the nucleotide sequence shown in SEQ ID NO.3, the downstream primer targets the nucleotide sequence shown in SEQ ID NO.4, and the probe is set to span the connecting region of SEQ ID NO.3 and SEQ ID NO.4; or, the upstream primer or the downstream primer targets the connecting region across SEQ ID NO.3 and SEQ ID NO.4.

[0076] Specifically, the specific targeting sequence of Salmonella paratyphi C is a nucleotide sequence as shown in SEQ ID NO.36, including the front part of the specific segment as shown in SEQ ID NO.5 and the rear part of the specific segment as shown in SEQ ID NO.6. In Salmonella paratyphi C, SEQ ID NO.5 and 6 directly constitute SEQ ID NO.36, and there is no nucleotide sequence in the connecting region, while in Salmonella paratyphi A and B, there are certain nucleotides between SEQ ID NO.5 and 6 as the connecting region. Designing amplification primers or probes across the connecting region of SEQ ID NO.5 and SEQ ID NO.6 can specifically detect Salmonella paratyphi C. For example, the upstream primer targets the nucleotide sequence shown in SEQ ID NO.5, the downstream primer targets the nucleotide sequence shown in SEQ ID NO.6, and the probe is set to span the connecting region of SEQ ID NO.5 and SEQ ID NO.6; or, the upstream primer or the downstream primer targets the connecting region across SEQ ID NO.5 and SEQ ID NO.6.

[0077] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0078] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0079] In a specific embodiment, the internal standard is RNase P or GAPDH.

[0080] In some specific embodiments, the upstream primer, downstream primer and probe for detecting the internal standard are shown in SEQ ID NOs. 37 to 39.

[0081] Furthermore, the fluorescent groups of the probes of the nucleic acid composition for detecting different types of Salmonella paratyphi are different from each other and do not interfere with each other.

[0082] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0083] In some specific embodiments, the fluorescent reporter group of the probe for detecting Salmonella Paratyphi A is FAM; the fluorescent reporter group of the probe for detecting Salmonella Paratyphi B is HEX; and the fluorescent reporter group of the probe for detecting Salmonella Paratyphi C is ROX.

[0084] Furthermore, in some embodiments, the composition of the present invention may simultaneously include one or more of the first nucleic acid composition, the second nucleic acid composition, and the third nucleic acid composition.

[0085] Those skilled in the art can combine as needed to detect which targets, that is, combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0086] For example, when detecting Salmonella Paratyphi A, the first nucleic acid composition may be included; when detecting Salmonella Paratyphi B, the second nucleic acid composition may be included; when detecting Salmonella Paratyphi C, the third nucleic acid composition may be included; when detecting Salmonella Paratyphi A and Salmonella B at the same time, the first and second nucleic acid compositions may be included; when detecting Salmonella Paratyphi A and Salmonella C at the same time, the first and third nucleic acid compositions may be included; when detecting Salmonella Paratyphi B and Salmonella C at the same time, the second and third nucleic acid compositions may be included; when detecting Salmonella Paratyphi A, B and C at the same time, the first, second and third nucleic acid compositions may be included.

[0087] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0088] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0089] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0090] In a specific embodiment, the quencher group at the 3' end of the probe is BHQ1.

[0091] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0092] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0093] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0094] In a second aspect, the present invention provides use of the composition of the present invention in preparing a kit for detecting and typing Salmonella Paratyphi, wherein the pathogen is one or more of Salmonella Paratyphi type A, B, and C.

[0095] In a third aspect, the present invention provides a kit for detecting and typing Salmonella Paratyphi, wherein the kit comprises the composition of the present invention as described above.

[0096] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0097] In a specific embodiment, the negative control product is at least one of DEPC H2O and normal saline; the positive control product is at least one of a fragment plasmid or a positive strain of Salmonella paratyphi type A, B, or C.

[0098] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0099] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG enzyme).

[0100] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0101] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG enzyme), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0102] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0103] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UNG enzyme, MgCl2, dNTP(U)s, primers, probe and PCR buffer.

[0104] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0105] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0106] In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0107] In a fourth aspect, a method for detecting and typing Salmonella paratyphi is provided, the method comprising the following steps:

[0108] 1) Extracting nucleic acid from the sample to be tested;

[0109] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0110] 3) Obtain and analyze the results.

[0111] In the present invention, the sample used for detection can be sputum, respiratory secretions, blood, etc., but is not limited thereto.

[0112] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0113] UNG enzyme reaction, temperature is 50-60℃, time is 0.5-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-95℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0114] Furthermore, the method for detecting and typing Salmonella Paratyphi is for non-diagnostic purposes.

[0115] In a specific embodiment, a composition is provided for preparing a reagent for detecting and typing Salmonella paratyphi, wherein the detection comprises the following steps:

[0116] 1) Extracting nucleic acid from the sample to be tested;

[0117] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0118] 3) Obtain and analyze the results.

[0119] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0120] UNG enzyme reaction, temperature is 50-60℃, time is 0.5-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-95℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0121] In this context, the term "non-diagnostic purpose" means that the method is not intended to obtain information on whether an individual is infected with the above pathogens and suffers from the infection. For example, the method can detect whether the environment, culture, and drinking water sources have the above pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 This is the detection diagram of different primer probe sets for Salmonella Paratyphi A;

[0123] Figure 2 This is the detection diagram of different primer probe sets for Salmonella Paratyphi B;

[0124] Figure 3 This is a diagram of the detection of different primer probe sets for Salmonella Paratyphi C;

[0125] Figure 4 The nucleic acid composition combination 1 is a detection chart of Salmonella paratyphi type A, B and C pathogens;

[0126] Figure 5 It is a nucleic acid composition combination 2 for detecting pathogens of Salmonella paratyphi type A, B and C;

[0127] Figure 6 It is a nucleic acid composition combination 3 pairs of Salmonella paratyphi pathogens A, B, C detection diagram;

[0128] Figure 7 This is the sensitivity test diagram of the primer-probe set for Salmonella Paratyphi A;

[0129] Figure 8 This is the sensitivity test diagram of the primer-probe set for Salmonella paratyphi B;

[0130] Fig. 9 This is the sensitivity test diagram of the primer-probe set for Salmonella Paratyphi C;

[0131] Fig.10 It is a specific detection diagram of nucleic acid composition combination 1;

[0132] Fig.11 This is a non-specific result diagram of the reagent in Comparative Example 1 for detecting Salmonella typhimurium in Bareilly;

[0133] Fig.12 This is a non-specific result diagram of the reagent combination for detecting Minnesota Salmonella in Comparative Example 2;

[0134] Fig.13 This is a non-specific result diagram of the reagent combination for detecting Salmonella cholera in Comparative Example 3;

[0135] Fig.14 This is a graph showing the non-specific results of the reagent for detecting type-A paratyphoid Salmonella to typhoid and cholera Salmonella in comparative example 4;

[0136] Fig.15 This is a graph showing the non-specific results of the reagent for detecting type B Salmonella paratyphi for comparative example 4 against Salmonella typhi and Salmonella cholera;

[0137] Fig.16 This is a graph showing the non-specific results of the reagent for detecting type C Salmonella paratyphi for Comparative Example 4 against Salmonella typhi and Salmonella cholera. DETAILED DESCRIPTION

[0138] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0139] Example 1: Primers and probes used in the present invention

[0140] The primers and probes used in the present invention are shown in Table 1 below:

[0141] Table 1

[0142]

[0143]

[0144]

[0145] Among them, SEQ ID NO 1 and SEQ ID NO 2 are continuous gene segments, which are respectively the front part and the rear part of the specific segment SEQ ID NO 34 of type Paratyphi Salmonella A; SEQ ID NO 3 and SEQ ID NO 4 are continuous gene segments, which are respectively the front part and the rear part of the specific segment SEQ ID NO 35 of type Paratyphi Salmonella B; SEQID NO 5 and SEQ ID NO 6 are continuous gene segments, which are respectively the front part and the rear part of the specific segment SEQID NO 36 of type Paratyphi Salmonella C.

[0146] The fluorescent reporter group of the probe for detecting type A Salmonella paratyphi is FAM; the fluorescent reporter group of the probe for detecting type B Salmonella paratyphi is HEX; the fluorescent reporter group of the probe for detecting type C Salmonella paratyphi is ROX; and the fluorescent reporter group of the probe for detecting the internal standard is CY5.

[0147] Example 2: Method for detecting pathogens using the composition of the present invention

[0148] Reagent preparation:

[0149] Preparation of enzyme mixture:

[0150] The enzyme mixture consists of UNG enzyme and H-Taq enzyme. H-Taq enzyme (15U / μL) and UNG enzyme (5U / μL) are mixed in a certain ratio (0.8μL H-Taq enzyme and 0.2μL UNG enzyme per person).

[0151] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture according to the proportion (PCR reaction solution 19 μL / person + enzyme mixture 1 μL / person), mix them thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds and set aside. The real-time fluorescence PCR reaction solution is configured according to the following Table 2.

[0152] Table 2

[0153] Components Volume / concentration in each reaction dNTPs(100mM) 0.50μL <![CDATA[1mol / L MgCl2]]> 0.20μL Primers 0.2μL (50pmol / uL) / strip Probe 0.1μL (50pmol / uL) / strip PCR Buffer Make up to 19 μL

[0154] Sample handling and loading:

[0155] Take 200 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. to extract nucleic acid according to its instructions; take 5 μL of the above-treated samples, negative controls, and positive controls and add them to the corresponding 0.2 mL PCR reaction tubes, add 20 μL of PCR mixture to each tube, and cover the tube.

[0156] PCR amplification:

[0157] PCR amplification was performed on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the temperature and time settings shown in Table 3.

[0158] Table 3

[0159]

[0160] Result analysis and judgment

[0161] If the sample has an obvious S-shaped amplification curve in the FAM, HEX, ROX and CY5 channels, and the Ct value is ≤40, it is judged as positive; if the sample has no amplification curve (No Ct) or the Ct value is greater than 40 in the FAM, HEX and ROX channels, and the CY5 internal standard channel is positive (Ct value ≤40), it is judged as negative. The details are shown in Table 4 below.

[0162] Table 4

[0163]

[0164]

[0165] Example 3: Test results of the test samples of the composition of the present invention

[0166] The primers and probes shown in Example 1 were used to perform PCR detection on single or mixed samples of type A, B, and C on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the method of Example 2. The detection results are shown in FIG. Figures 1 to 6As shown in the figure, it can be seen that the composition of the present invention can perform good detection on various pathogens alone or in combination:

[0167] The first nucleic acid composition (respectively: 1. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.7, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.10, and a probe having a nucleotide sequence as shown in SEQ ID NO.25; 2. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.8, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.11, and a probe having a nucleotide sequence as shown in SEQ ID NO.26; 3. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.9, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.12, and a probe having a nucleotide sequence as shown in SEQ ID NO.27) Figure 1 );

[0168] The second nucleic acid composition (respectively: 1. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.13, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.16, and a probe having a nucleotide sequence as shown in SEQ ID NO.28; 2. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.14, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.17, and a probe having a nucleotide sequence as shown in SEQ ID NO.29; 3. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.15, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.18, and a probe having a nucleotide sequence as shown in SEQ ID NO.30) Figure 2 );

[0169] The third nucleic acid composition (respectively: 1. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.19, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.22, and a probe having a nucleotide sequence as shown in SEQ ID NO.31; 2. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.20, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.23, and a probe having a nucleotide sequence as shown in SEQ ID NO.32; 3. an upstream primer having a nucleotide sequence as shown in SEQ ID NO.21, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.24, and a probe having a nucleotide sequence as shown in SEQ ID NO.33) Figure 3 );

[0170] from Figures 1 to 3It can be seen that each set of primers and probes in each nucleic acid composition can detect the corresponding target.

[0171] Nucleic acid composition combination 1 (comprising: an upstream primer having a nucleotide sequence as shown in SEQ ID NO.7, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.10, and a probe having a nucleotide sequence as shown in SEQ ID NO.25; an upstream primer having a nucleotide sequence as shown in SEQ ID NO.13, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.16, and a probe having a nucleotide sequence as shown in SEQ ID NO.28; an upstream primer having a nucleotide sequence as shown in SEQ ID NO.19, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.22, and a probe having a nucleotide sequence as shown in SEQ ID NO.31) Figure 4 );

[0172] Nucleic acid composition combination 2 (comprising: an upstream primer having a nucleotide sequence as shown in SEQ ID NO.8, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.11, and a probe having a nucleotide sequence as shown in SEQ ID NO.26; an upstream primer having a nucleotide sequence as shown in SEQ ID NO.14, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.17, and a probe having a nucleotide sequence as shown in SEQ ID NO.29; an upstream primer having a nucleotide sequence as shown in SEQ ID NO.20, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.23, and a probe having a nucleotide sequence as shown in SEQ ID NO.32) ( Figure 5 )

[0173] Nucleic acid composition combination 3 (comprising: an upstream primer having a nucleotide sequence as shown in SEQ ID NO.9, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.12, and a probe having a nucleotide sequence as shown in SEQ ID NO.27; an upstream primer having a nucleotide sequence as shown in SEQ ID NO.15, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.18, and a probe having a nucleotide sequence as shown in SEQ ID NO.30; an upstream primer having a nucleotide sequence as shown in SEQ ID NO.21, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.24, and a probe having a nucleotide sequence as shown in SEQ ID NO.33) Figure 6 )

[0174] from Figures 4 to 6 It can be seen that each combination of nucleic acid compositions can detect the corresponding combination of targets.

[0175] Example 4: Sensitivity of the composition of the present invention

[0176] Using the nucleic acid composition combination 1 in Example 3 of the present invention, gradient dilution plasmids were taken, and the concentrations were 500000, 50000, 5000, and 500 copies / mL, respectively, and 4 concentrations of Salmonella Paratyphi A plasmid, Salmonella Paratyphi B plasmid, and Salmonella Paratyphi C plasmid were detected respectively.

[0177] The test results of the 500 copies / mL sample are as follows Figure 7 , 8 As shown in Figures 9 and Table 5, it is shown that each channel can still accurately detect samples as low as 500 copies / mL, with a detection rate of 100%, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0178] Table 5

[0179] Pathogen type Detection number Detection rate% Salmonella Paratyphi A 8 100 Salmonella Paratyphi B 8 100 Salmonella Paratyphi C 8 100

[0180] Example 5. Specificity of the composition of the present invention

[0181] The composition of the present invention (including Figures 1 to 6 The combination shown in the table had no cross-reaction with other common pathogens (Staphylococcus aureus, Salmonella Typhi, Salmonella Typhimurium Bareilly, Salmonella Minnesota, Salmonella Cholera, Bacteroides fragilis, Helicobacter pylori, Streptococcus pneumoniae, Vibrio parahaemolyticus, Shigella, and diarrheagenic Escherichia coli). Fig.10 As shown, it is shown that the composition of the present invention has good specificity.

[0182] Comparative Example 1

[0183] Inoculate the standard strain of Salmonella typhimurium (enterica serovar Bareilly, standard product number: ATCC9115) into 50 ml of LB liquid culture medium. After culturing at 37°C for 8 hours, take 1 ml of the bacterial solution and put it into a centrifuge tube. Centrifuge at 3000r / min for 10 minutes, take the supernatant, and centrifuge at 12000r / min for 5 minutes to collect the cell pellet. Resuspend the pellet with sterile deionized water. After washing, add 100μL of sterile ultrapure water. Boil in a boiling water bath for 15 minutes, take out immediately and place in a -20°C environment for 30 minutes. After thawing at 37°C, centrifuge again at 12,000r / min for 5 minutes, take the supernatant, and place it at -20°C for use. The extracted Salmonella typhimurium DNA solution was mixed with the internal standard solution in a ratio of 1:1, and 5 μL of the sample solution was taken according to the method of Example 2, and the primer combination of the prior art (CN102634577A) was used for detection by a Hongshi fluorescent quantitative PCR instrument. The detection results are as follows: Fig.11The prior art comparative reagent (CN102634577A) tested positive for Salmonella typhimurium (Serovar Bareilly), but could not distinguish between the bacteria and Paratyphoid A.

[0184] Comparative Example 2

[0185] The primer-probe combination of the disclosed technical solution (CN102102124A) and the method of Example 2 was used for detection. The standard strain enrichment and extraction process refer to Comparative Example 1. The Salmonella enterica Minnesota DNA solution was mixed with the internal standard solution in a ratio of 1:1. 5 μL of the sample solution was taken and tested by a Macrostone fluorescent quantitative PCR instrument according to the method of Example 2. The test results are shown in the figure. Fig.12 The comparative reagent tested positive for Salmonella serovar Minnesota and could not distinguish between the bacteria and Paratyphoid B.

[0186] Comparative Example 3

[0187] The primer-probe combination of the disclosed technical solution (DOI: 10.19813 / j.cnki.weishengyanjiu.2017.02.024) was used to detect Salmonella cholerae (enterica serovar Choleraesuis, standard product number: ATCC 10708) using the method of Example 2. The standard strain enrichment and extraction process refer to Comparative Example 1. The Salmonella cholerae DNA solution was mixed with the internal standard solution in a ratio of 1:1. 5 μL of the sample solution was taken and tested using the Macrostone fluorescent quantitative PCR instrument according to the method of Example 2. The test results are shown in FIG. Fig.13 The comparative reagent tested positive for Salmonella cholerae (enterica serovar Choleraesuis) and could not distinguish between the bacterium and Paratyphoid C.

[0188] Comparative Example 4

[0189] The present invention screens out the conservative and specific target regions of the target species through whole genome comparative analysis, and the primer and probe combination used for detection needs to cover the differential segment. The inventors designed different detection systems composed of other primers and probes near the target region (the sequence is not shown, specifically, the primers / probes are set in the connecting region that does not cross the front and rear parts of the specific segment of the target species, that is, the upstream and downstream primers and probes are set in the front or rear part of the specific segment), which are also used to detect Salmonella paratyphi and other Salmonella (Salmonella typhi, Salmonella cholera).

[0190] Among them, the upstream and downstream primers for detecting Salmonella paratyphi A both target SEQ ID NO 1;

[0191] The upstream and downstream primers for detecting Salmonella paratyphi B both target SEQ ID NO 4;

[0192] The upstream and downstream primers for detecting Salmonella Paratyphi C both target SEQ ID NO 5.

[0193] Paratyphoid, typhoid and cholera Salmonella samples were added to three different PCR reaction tubes and placed on the Macrostone fluorescent quantitative PCR instrument for PCR detection. The specific test results are as follows: Fig.14 , 15 , as shown in 16.

[0194] from Fig.14 It can be seen that the other primer probes for detecting Salmonella Paratyphi A were positive for Salmonella Typhi and Salmonella Cholera;

[0195] from Fig.15 It can be seen that the other primer probes for detecting Salmonella Paratyphi B were positive for Salmonella Typhi and Salmonella Cholera;

[0196] from Fig.16 It can be seen that the other primer probes for detecting S. paratyphi C tested positive for S. typhi and S. cholerae;

[0197] It can be seen from this that for the design of upstream and downstream primers and probes for Salmonella paratyphi A, B and C, when the upstream and downstream primers and probes are simultaneously set in the front part or the back part of the specific segment, that is, when the specific conserved connecting region is not detected, the primer-probe system combination will show non-specific amplification in the detection of Salmonella typhi and Salmonella cholera, and the serotype cannot be accurately distinguished.

[0198] Other detection system combinations near the target area were positive for Salmonella paratyphi, Salmonella typhi and Salmonella cholera, but were unable to distinguish the serotypes.

Claims

1. A composition for detecting and typing Salmonella paratyphi, comprising at least one of the following nucleic acid compositions: A first nucleic acid composition comprising: 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.1, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.2, and a probe arranged across the junction region between SEQ ID NO.1 and SEQ ID NO.2; or, 2) an upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO.1 and SEQ ID NO.2; A second nucleic acid composition comprising: 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.3, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.4, and a probe arranged across the junction region of SEQ ID NO.3 and SEQ ID NO.4; or, 2) an upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO. 3 and SEQ ID NO. 4; or A third nucleic acid composition comprises: 1) an upstream primer targeting the nucleotide sequence shown in SEQ ID NO.5, a downstream primer targeting the nucleotide sequence shown in SEQ ID NO.6, and a probe arranged across the junction region of SEQ ID NO.5 and SEQ ID NO.6; or, 2) An upstream primer or a downstream primer targeting the junction region spanning SEQ ID NO. 5 and SEQ ID NO.

6.

2. The composition according to claim 1, characterized in that The upstream primer in the first nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 7 to 9; The downstream primer in the first nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 10 to 12; The probe of the first nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 25-27.

3. The composition according to claim 1, characterized in that The upstream primer in the second nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 13 to 15; The downstream primer in the second nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 16 to 18; The probe of the second nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 28-30.

4. The composition according to claim 1, characterized in that The upstream primer in the third nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 19 to 21; The downstream primer in the third nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 22 to 24; The probe of the third nucleic acid composition includes at least one of the nucleotide sequences shown in SEQ ID NOs. 31-33.

5. The composition according to claim 1, characterized in that The composition comprises at least two or three of any of the above nucleic acid compositions.

6. The composition according to claim 1, characterized in that The composition comprises: A first nucleic acid composition comprising: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.7, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.10, and a probe having a nucleotide sequence as shown in SEQ ID NO.25; A second nucleic acid composition comprising: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.13, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.16, and a probe having a nucleotide sequence as shown in SEQ ID NO.28; and A third nucleic acid composition comprises: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.19, an upstream primer having a nucleotide sequence as shown in SEQ ID NO.22, and a probe having a nucleotide sequence as shown in SEQ ID NO.31; or A first nucleic acid composition comprising: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.8, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.11, and a probe having a nucleotide sequence as shown in SEQ ID NO.26; A second nucleic acid composition comprising: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.14, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.17, and a probe having a nucleotide sequence as shown in SEQ ID NO.29; A third nucleic acid composition comprises: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.20, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.23, and a probe having a nucleotide sequence as shown in SEQ ID NO.32; or A first nucleic acid composition comprising: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.9, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.12, and a probe having a nucleotide sequence as shown in SEQ ID NO.27; A second nucleic acid composition comprising: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.15, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.18, and a probe having a nucleotide sequence as shown in SEQ ID NO.30; A third nucleic acid composition comprises: The nucleotide sequence of the upstream primer is shown as SEQ ID NO.21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.24, and the nucleotide sequence of the probe is shown as SEQ ID NO.

33.

7. Use of the composition according to any one of claims 1 to 6 in preparing a kit for detecting and typing Salmonella paratyphi, wherein The paratyphoid Salmonella includes one or more of paratyphoid Salmonella types A, B and C.

8. A kit for detecting and typing Salmonella Paratyphi, comprising the composition according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit further comprises: a nucleic acid releasing reagent, a nucleic acid extracting reagent, a DNA polymerase, a UNG enzyme, dNTP, dUTP, a UNG enzyme, a PCR buffer, or a Mg 2+ At least one of .

10. Use of a composition for preparing a reagent for detecting and typing Salmonella paratyphi, wherein the detection comprises the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing fluorescent PCR detection on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to any one of claims 8 to 9; 3) Obtain and analyze the results.

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