Composition for detecting intestinal infection related pathogens, kit and application

Abstract

The invention belongs to the field of molecular biological detection, particularly relates to detection of intestinal infection related pathogens, and more particularly relates to detection of at least four of salmonella typhimurium, salmonella paratyphi, rickettsia, pathogenic leptospirosis and plasmodium. The composition provided by the invention has the advantages of higher detection sensitivity (up to 500 copies / mL), good specificity and more accurate detection, provides a more sufficient basis for rapid diagnosis and exclusion of infection of different pathogens for clinicians, shortens the time for the clinicians to diagnose the condition of a patient, and accelerates the implementation of treatment measures on the patient.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of pathogens related to intestinal infection, and more specifically, to the detection of Salmonella typhi, Salmonella paratyphi, Rickettsia, pathogenic Leptospira, and Plasmodium. Background Art

[0002] Salmonella enterica serovar Typhi includes the enteric subspecies Typhimurium. It is Gram-negative, short and thick rod-shaped, covered with flagella, and active in movement. It is the pathogen that causes typhoid fever. Humans are its only natural host. The disease often develops gradually, with symptoms such as fever, headache, joint pain, pharyngitis, constipation, poor appetite, abdominal pain and tenderness. Dysuria, dry cough and epistaxis are less common, and complications include intestinal bleeding and intestinal perforation.

[0003] Salmonella enterica serotypes include enteric subspecies paratyphi A, B, or C. Under natural conditions, Salmonella enterica serotypes paratyphi generally only infect humans and occasionally animals, and are the pathogens that cause paratyphoid fever. The symptoms of paratyphoid A and B are similar to those of typhoid fever, but the disease is generally milder, the course is shorter, and the mortality rate is lower. The symptoms of paratyphoid C are quite different and can manifest as mild typhoid fever, acute gastroenteritis, or sepsis.

[0004] Rickettsia is a type of prokaryotic microorganism that is strictly parasitic in living cells. Its biological activity is close to that of bacteria. It has no nucleolus and nuclear membrane and is generally spherical or rod-shaped. Common rickettsia include Rickettsia prowazekii, Rickettsia morozei, Rickettsia rickettsii and Rickettsia tsutsugamushi, which can also cause various typhoid fevers. The clinical symptoms mainly include high fever, headache, rash and central nervous system symptoms. Leptospira belongs to the family of the order Treponema. The bacteria are slender, of varying lengths, with fine and regular spirals, and one or both ends of the bacteria are bent into a hook shape. Humans can be directly infected by contact with the urine or tissue of infected animals, or indirectly infected by contact with contaminated water or soil. The clinical manifestations vary. In the early stage, they mainly manifest as chills, fever, headache, fatigue, conjunctival congestion, muscle pain, etc. If not treated in time, symptoms such as hemoptysis, pulmonary hemorrhage, jaundice, hypotension, shock, renal failure, and meningoencephalitis may occur. Severe cases may be life-threatening.

[0005] Plasmodium is a group of single-celled, parasitic protozoa. Organisms in this genus are collectively known as Plasmodium. There are five species of Plasmodium in this genus that infect humans with malaria through the bite of female mosquitoes: Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi. Malaria can cause fever, chills, sweating, a general malaise, and sometimes diarrhea, abdominal pain, respiratory distress, confusion, and seizures. Other findings include an enlarged spleen, anemia, and sometimes damage to the heart, brain, lungs, or kidneys.

[0006] The above pathogens have similar clinical manifestations in the early stages of the disease, causing fever, headache, chills, muscle aches and other symptoms. For infected patients, early diagnosis is needed to identify the cause of infection, early treatment, and timely control of disease progression, blocking the transmission route, and preventing further spread of the disease. At present, the diagnosis of the above pathogens mainly uses bacterial culture and serological reactions, which have problems such as low sensitivity and long time consumption.

[0007] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens, and has good sensitivity and specificity, and can detect and distinguish pathogenic microorganisms that cause infection, so as to provide targeted strategies for the prevention and treatment of the above-mentioned infections. Summary of the invention

[0008] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens related to intestinal infection, comprising at least 4 of the following nucleic acid compositions:

[0009] A first nucleic acid composition comprising:

[0010] Upstream primers, downstream primers and probes for detecting Salmonella typhi as shown in SEQ ID NOs: 1 to 3;

[0011] The second nucleic acid composition includes upstream and downstream primers and probes for detecting at least one of the following related pathogens:

[0012] Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi A as shown in SEQ ID NOs: 4 to 6;

[0013] The upstream primers, downstream primers and probes for detecting Salmonella paratyphi B as shown in SEQ ID NOs: 7 to 9; or

[0014] Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi C as shown in SEQ ID NOs: 10 to 12;

[0015] A third nucleic acid composition comprises:

[0016] Upstream primers, downstream primers and probes for detecting Rickettsia as shown in SEQ ID NOs: 13 to 15;

[0017] A fourth nucleic acid composition comprising:

[0018] Upstream primers, downstream primers and probes for detecting pathogenic Leptospira as shown in SEQ ID NOs: 16 to 18; or,

[0019] The fifth nucleic acid composition comprises:

[0020] The upstream primers, downstream primers and probe for detecting Plasmodium are shown in SEQ ID NOs: 19-21.

[0021] The combined detection composition provided by the present invention mainly utilizes a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of at least 4 of Salmonella typhi, Salmonella paratyphi, Rickettsia, pathogenic Leptospira, and Plasmodium in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. The composition of the present invention has a higher detection sensitivity of 500 copies / mL, good specificity, and more accurate detection, providing clinicians with a relatively sufficient basis for rapid diagnosis and exclusion of different pathogen infections, shortening the time for clinicians to diagnose the patient's condition, and accelerating the implementation of treatment measures for the patient.

[0022] Furthermore, the present invention provides a composition for detecting pathogens related to intestinal infection, comprising the following nucleic acid composition:

[0023] A first nucleic acid composition comprising:

[0024] Upstream primers, downstream primers and probes for detecting Salmonella typhi as shown in SEQ ID NOs: 1 to 3;

[0025] The second nucleic acid composition includes upstream and downstream primers and probes for detecting at least one of the following related pathogens:

[0026] Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi A as shown in SEQ ID NOs: 4 to 6;

[0027] The upstream primers, downstream primers and probes for detecting Salmonella paratyphi B as shown in SEQ ID NOs: 7 to 9; or

[0028] Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi C as shown in SEQ ID NOs: 10 to 12;

[0029] A third nucleic acid composition comprises:

[0030] Upstream primers, downstream primers and probes for detecting Rickettsia as shown in SEQ ID NOs: 13 to 15;

[0031] A fourth nucleic acid composition comprising:

[0032] Upstream primers, downstream primers and probes for detecting pathogenic Leptospira as shown in SEQ ID NOs: 16 to 18; and

[0033] A fifth nucleic acid composition comprising:

[0034] The upstream primers, downstream primers and probe for detecting Plasmodium are shown in SEQ ID NOs: 19-21.

[0035] Furthermore, the second nucleic acid composition includes upstream and downstream primers and probes for detecting at least two of the following related pathogens:

[0036] Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi A as shown in SEQ ID NOs: 4 to 6;

[0037] The upstream primers, downstream primers and probes for detecting Salmonella paratyphi B as shown in SEQ ID NOs: 7 to 9; or

[0038] The upstream primers, downstream primers and probe for detecting Salmonella Paratyphi C are shown in SEQ ID NOs: 10-12.

[0039] Furthermore, the second nucleic acid composition comprises:

[0040] Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi A as shown in SEQ ID NOs: 4 to 6;

[0041] Upstream primers, downstream primers and probes for detecting Salmonella paratyphi B as shown in SEQ ID NOs: 7 to 9; and

[0042] The upstream primers, downstream primers and probe for detecting Salmonella Paratyphi C are shown in SEQ ID NOs: 10-12.

[0043] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0044] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0045] In a specific embodiment, the internal standard is RNase P or NADPH.

[0046] Furthermore, the fluorescent groups of the probes used in the composition of the present invention to detect different pathogens are different from each other and do not interfere with each other.

[0047] 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.

[0048] In some specific embodiments, the fluorescent reporter group of the probe for detecting Salmonella typhi is FAM; the fluorescent reporter group of the probe for detecting Salmonella Paratyphi A, Salmonella Paratyphi B, and Salmonella Paratyphi C is HEX; the fluorescent reporter group of the probe for detecting Rickettsia is ROX; the fluorescent reporter group of the probe for detecting pathogenic Leptospira is CY5; and the fluorescent reporter group of the probe for detecting Plasmodium is QUASAR 705.

[0049] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0050] The compositions of the present invention can be combined into any combination of the corresponding five nucleic acid compositions. Those skilled in the art can combine as needed to detect which nucleic acid compositions to detect, that is, to combine the primers and probe pairs of the corresponding nucleic acid compositions. These combinations are all included in the present invention.

[0051] The composition of the present invention can be combined into any combination of detection corresponding to the 7 targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0052] For example, it may include any 6 of the above 7 sets of primers and probes, any 5 of the above 7 sets of primers and probes, any 4 of the above 7 sets of primers and probes, or any 3 of the above 7 sets of primers and probes.

[0053] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0054] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0055] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0056] In a specific embodiment, the 3' end of the probe is BHQ1.

[0057] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0058] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0059] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0060] In a second aspect, the present invention provides a use of the above-mentioned composition of the present invention in the preparation of a kit for detecting intestinal infection-related pathogens, wherein the pathogen is one or more of pathogenic Leptospira, Salmonella typhi, Salmonella paratyphi, Rickettsia, or Plasmodium.

[0061] In a third aspect, the present invention provides a kit for detecting pathogens associated with intestinal infection, wherein the kit comprises the composition of the present invention as described above.

[0062] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0063] In a specific embodiment, the negative control is DEPC H 2 O, at least one of normal saline; the positive quality control product is at least one of the fragment plasmid or positive strain of pathogenic Leptospira, Salmonella typhi, Salmonella paratyphi (type A, B, C), Rickettsia, and Plasmodium.

[0064] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0065] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0066] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0067] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG), DNA polymerase, reverse transcriptase, PCR buffer and Mg 2+ At least one of .

[0068] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0069] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UDG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0070] The main components of PCR buffer include Tris-HCl, NaCl, NH 4 Cl, Tween 20.

[0071] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0072] 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.

[0073] In a fourth aspect, a method for detecting intestinal infection-related pathogens is provided, the method comprising the following steps:

[0074] 1) Extracting nucleic acid from the sample to be tested;

[0075] 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);

[0076] 3) Obtain and analyze the results.

[0077] In the present invention, the sample used for detection may be blood, etc., but is not limited thereto.

[0078] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0079] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0080] Furthermore, the method for detecting intestinal infection-related pathogens is for non-diagnostic purposes.

[0081] In a specific embodiment, a composition is provided for preparing a reagent for detecting intestinal infection-related pathogens, wherein the detection comprises the following steps:

[0082] 1) Extracting nucleic acid from the sample to be tested;

[0083] 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);

[0084] 3) Obtain and analyze the results.

[0085] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0086] Reverse transcription, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence collection.

[0087] 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

[0088] Figures 1 to 3 The results of testing different samples for the composition of the present invention are shown in FIG.

[0089] Figures 4 to 8 The sensitivity result diagram of the composition of the present invention (Salmonella typhi, Salmonella paratyphi (type A, B, C), Rickettsia, pathogenic Leptospira, Plasmodium);

[0090] Fig. 9 It is a specific result diagram of the composition of the present invention;

[0091] Figures 10 to 14 The anti-interference result diagram of the composition of the present invention (Salmonella typhi, Salmonella paratyphi (type A, B, C), Rickettsia, pathogenic Leptospira, Plasmodium);

[0092] Figures 15 to 19 The precision result diagram of the composition of the present invention (Salmonella typhi, Salmonella paratyphi (type A, B, C), Rickettsia, pathogenic Leptospira, Plasmodium) respectively;

[0093] Figures 20-22 This is a graph showing the test results of different compositions of the comparative examples of the present invention. DETAILED DESCRIPTION

[0094] 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.

[0095] Example 1: Primers and probes used in the present invention

[0096] The primers and probes used in the present invention are shown in Table 1 below:

[0097] Table 1

[0098]

[0099] Among them, the fluorescent reporter group of the Salmonella typhi probe is FAM; the fluorescent reporter group of the Salmonella Paratyphi A, Salmonella Paratyphi B and Salmonella Paratyphi C probes is HEX; the fluorescent reporter group of Rickettsia is ROX; the fluorescent reporter group of the pathogenic Leptospira probe is CY5; and the fluorescent reporter group of the probe for detecting Plasmodium is QUASAR 705.

[0100] Example 2: Method for detecting pathogens

[0101] Reagent preparation

[0102] Take out each component in the box, place it at room temperature, wait for the temperature to equilibrate to room temperature, mix well and centrifuge for a short time before use.

[0103] According to the number of samples to be tested, negative controls, and positive controls, take the corresponding amount of components in proportion (26 μL / person of PCR reaction solution + 4 μL / person of enzyme mixture), mix them thoroughly to form a PCR mixture, and centrifuge it for standby use.

[0104] Transfer the above prepared reagents to the sample processing area for later use.

[0105] Sample handling and loading

[0106] The test sample of the present invention is blood. The viral nucleic acid is extracted by magnetic bead method, and the following operations are performed in the sample processing room:

[0107] Take a 1.5mL cryotube and add glass beads. Add 300uL of the treated sample and 300uLTE to the 1.5mL cryotube containing mixed glass beads and grind using a grinder. When the grinding is completed, take out the cryotube and centrifuge it instantly. Take out the supernatant liquid as the sample to be tested for later use.

[0108] Take a number of 1.5mL centrifuge tubes according to the number of samples to be tested, and add 300μL of the sample to be tested to each tube;

[0109] Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cover the tube, shake and mix for 30 seconds, and heat at 60°C for 10 minutes.

[0110] Let stand at room temperature for 1 min, centrifuge at low speed, place the centrifuge tube on the magnetic separator, and slowly aspirate and discard the waste liquid after 5 min (*Be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall);

[0111] Add 600 μL of washing solution 1, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0112] Add 600 μL of washing solution 2, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0113] Place the centrifuge tube in a centrifuge and centrifuge it at low speed for a short time, then place the centrifuge tube in a magnetic separator again. Magnetic suction for 3 minutes to completely absorb the liquid at the bottom of the tube.

[0114] Add 30-100 μL of elution buffer S (80 μL is recommended), shake and mix for 30 seconds to elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, and let it stand at room temperature for 3 minutes; centrifuge at a low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0115] 20 μL of the treated samples, positive controls and negative controls were pipetted into the corresponding 0.2 mL PCR reaction tubes, 30 μL of PCR mixture was added to each tube, and the tube cap was closed. The real-time fluorescence PCR reaction system was configured according to Table 2 below.

[0116] Table 2

[0117] Components Volume / concentration in each reaction PCR buffer 22.3μL <![CDATA[MgCl 2 Solution (1mol / L)]]> 0.2μL dNTPs(U) 1.0μL Taq enzyme (5U / mL) 3μL RT enzyme (5U / mL) 1μL Primers 0.2μL (50pmol / μL) Probe 0.1μL (50pmol / μL)

[0118] PCR amplification

[0119] PCR amplification was performed on PCR instruments such as ABI 7500 fluorescence quantitative PCR instrument and SLAN-96P fully automatic medical PCR analysis system according to certain temperature and time setting programs. PCR amplification was performed according to the temperature and time setting programs shown in Table 3.

[0120] Table 3

[0121]

[0122] Result analysis and judgment

[0123] If the sample has an obvious S-shaped amplification curve in the FAM, HEX (VIC), ROX, CY5, and QUASAR 705 channels and the Ct value is ≤40, it is judged as positive; if the sample has no amplification curve (NoCt) or the Ct value is greater than 40 in the FAM, HEX (VIC), ROX, CY5, and Quasar 705 channels, it is judged as negative.

[0124] 1. For samples with a typical S-shaped amplification curve detected in the FAM channel and Ct≤40, it is reported as positive for Salmonella Typhi; for samples with no typical S-shaped amplification curve detected in the FAM channel, or Ct>40, it is reported as negative for Salmonella Typhi.

[0125] 2. For samples with a typical S-shaped amplification curve detected in the HEX channel and Ct≤40, it is reported as positive for Salmonella Paratyphi; for samples with no typical S-shaped amplification curve detected in the HEX channel, or Ct>40, it is reported as negative for Salmonella Paratyphi.

[0126] 3. For samples with a typical S-shaped amplification curve detected in the ROX channel and Ct≤40, it is reported as Rickettsia positive; for samples with no typical S-shaped amplification curve detected in the ROX channel, or Ct>40, it is reported as Rickettsia negative.

[0127] 4. For samples with a typical S-shaped amplification curve detected in the CY5 channel and Ct≤40, it is reported as positive for pathogenic Leptospira; for samples with no typical S-shaped amplification curve detected in the CY5 channel, or Ct>40, it is reported as negative for pathogenic Leptospira.

[0128] 5. For samples with a typical S-type amplification curve detected by QUASAR 705 channel and Ct≤40, it is reported as Plasmodium positive; for samples with no typical S-type amplification curve detected by QUASAR 705 channel, or Ct>40, it is reported as Plasmodium negative. The details are shown in Table 4 below.

[0129] Table 4

[0130]

[0131] Example 3: Test results of the test samples of the composition of the present invention

[0132] The primers and probes shown in Example 1 were used to perform PCR detection on a mixed sample of Salmonella typhi, Salmonella paratyphi (including types A, B, and C), Rickettsia, and pathogenic Leptospira on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the method of Example 2. The results showed that the composition of the present invention can detect and distinguish any combination of the above-mentioned related pathogens. For example, the composition of the present invention detects:

[0133] Paratyphoid Salmonella (A), Rickettsia, Pathogenic Leptospira, Plasmodium ( Figure 1 );

[0134] Typhoid Salmonella, Rickettsia, Pathogenic Leptospira, Plasmodium ( Figure 2 );

[0135] Salmonella typhi, Salmonella paratyphi (including types A, B, and C), Rickettsia, pathogenic Leptospira, Plasmodium ( Figure 3 ).

[0136] These figures prove that the present invention is a 5-plex PCR that can amplify the above 5 targets at one time, and can be distinguished using a 5-channel PCR instrument. If an 8-channel PCR instrument can be used, the composition of the present invention can also distinguish between types A, B, and C of Salmonella paratyphi (using different fluorescent groups). It also proves that any corresponding pair of combinations of the composition of the present invention can detect any corresponding combination of pathogens.

[0137] Example 4: Sensitivity of the composition of the present invention

[0138] Using the composition in Example 1 of the present invention, a gradient dilution of the plasmid was taken, and the concentrations were 500000, 50000, 5000, and 500 copies / mL, respectively, and the five concentrations were tested. Figures 4 to 8 As shown, 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.

[0139] Example 5. Specificity of the composition of the present invention

[0140] The composition of the present invention has no cross-reaction with other pathogens similar to intestinal infection-related pathogens (Vibrio parahaemolyticus, Yersinia enterica, Staphylococcus aureus, Proteus, Shigella flexneri, Listeria monocytogenes, enteropathogenic Escherichia coli Shigella dysenteriae, Streptococcus pneumoniae, epidemic hemorrhagic fever virus, Hantavirus and poliovirus). Fig. 9 As shown, it is shown that the composition of the present invention has good specificity.

[0141] Example 6: Anti-interference performance of the composition of the present invention

[0142] The composition of Example 1 was analyzed for the addition of interfering substances. The experimental results showed that potential PCR inhibitors / interfering substances such as 20 μg / mL mucin, 0.5 mg / mL meropenem, 20 μg / mL trimethoprim, 0.44 mg / mL sulfamethoxazole, 0.8 mg / mL amphotericin B, 0.1 g / mL itraconazole, 0.5 mg / mL fluconazole, 0.1 g / ml azithromycin, and 4 mg / mL lidocaine hydrochloride had no significant effect on the kit. The results are shown in Figures 10 to 14.

[0143] Example 7: Precision of the composition of the present invention

[0144] The system of the present invention dilutes the positive samples to two concentration levels: medium concentration positive (R1) and critical concentration positive (R2) (5.0×10 5 copies / mL and 5.0×10 3The intra-batch and inter-batch precisions were determined by 100% detection rate of strong positive and weak positive reference products, and the coefficient of variation (CV) of the intra-batch and inter-batch Ct values ​​was less than 5%, indicating good detection precision. Figures 15 to 19 shown.

[0145] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0146] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. In order to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0147] Therefore, the inventors also designed other primers and probes to form different detection systems (sequences are shown in Table 5), which are also used to detect the above pathogens. Figures 20-22 As shown in the figure, it can be seen that the test results are normal under the two / three pathogens combined detection system, all target amplification curves appear, the curve amplification is high, and the repeatability is good. Fig. 20 , Fig.21 However, only some of the amplification curves of the five pathogens were delayed in CT, and the amplification curves had low amplitude and poor repeatability, such as Fig. 22 As shown, the overall detection effect is poor.

[0148] Table 5

[0149]

Claims

1. A composition for detecting pathogens associated with intestinal infection, comprising at least 4 of the following nucleic acid compositions: A first nucleic acid composition comprising: Upstream primers, downstream primers and probes for detecting Salmonella typhi as shown in SEQ ID NOs: 1 to 3; The second nucleic acid composition includes upstream and downstream primers and probes for detecting at least one of the following related pathogens: Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi A as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Salmonella paratyphi B as shown in SEQ ID NOs: 7 to 9; or Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi C as shown in SEQ ID NOs: 10 to 12; A third nucleic acid composition comprises: Upstream primers, downstream primers and probes for detecting Rickettsia as shown in SEQ ID NOs: 13 to 15; A fourth nucleic acid composition comprising: Upstream primers, downstream primers and probes for detecting pathogenic Leptospira as shown in SEQ ID NOs: 16 to 18; or, The fifth nucleic acid composition comprises: The upstream primers, downstream primers and probe for detecting Plasmodium are shown in SEQ ID NOs: 19-21.

2. The composition according to claim 1, characterized in that The composition includes the following nucleic acid composition: A first nucleic acid composition comprising: Upstream primers, downstream primers and probes for detecting Salmonella typhi as shown in SEQ ID NOs: 1 to 3; The second nucleic acid composition includes upstream and downstream primers and probes for detecting at least one of the following related pathogens: Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi A as shown in SEQ ID NOs: 4 to 6; The upstream primers, downstream primers and probes for detecting Salmonella paratyphi B as shown in SEQ ID NOs: 7 to 9; or Upstream primers, downstream primers and probes for detecting Salmonella Paratyphi C as shown in SEQ ID NOs: 10 to 12; A third nucleic acid composition comprises: Upstream primers, downstream primers and probes for detecting Rickettsia as shown in SEQ ID NOs: 13 to 15; A fourth nucleic acid composition comprising: Upstream primers, downstream primers and probes for detecting pathogenic Leptospira as shown in SEQ ID NOs: 16 to 18; and The fifth nucleic acid composition comprises: The upstream primers, downstream primers and probe for detecting Plasmodium are shown in SEQ ID NOs: 19-21.

3. The composition according to claim 2, characterized in that The fluorescent reporter group of the probe for detecting Salmonella typhi is FAM; the fluorescent reporter group of the probe for detecting Salmonella paratyphi A, Salmonella paratyphi B and / or Salmonella paratyphi C is HEX; the fluorescent reporter group of the probe for detecting Rickettsia is ROX; the fluorescent reporter group of the probe for detecting pathogenic Leptospira is CY5; and the fluorescent reporter group of the probe for detecting Plasmodium is QUASAR 705.

4. The composition according to any one of claims 1 to 3, characterized in that The composition also includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

5. The composition according to claim 4, characterized in that The components of the composition are present in admixture.

6. Use of the composition according to any one of claims 1 to 5 in preparing a kit for detecting pathogens related to intestinal infection, wherein: The pathogen is one or more of pathogenic Leptospira, Salmonella typhi, Salmonella paratyphi, Rickettsia, or Plasmodium.

7. A kit for detecting pathogens associated with intestinal infection, the kit comprising the composition according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to claim 7 or 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a DNA polymerase, dNTP, dUTP, UNG enzyme, a PCR buffer, or Mg 2+ At least one of .

10. Use of a composition for preparing a reagent for detecting pathogens related to intestinal infection, the detection comprising 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 5 or the kit according to any one of claims 7 to 9; 3) Obtain and analyze the results.

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