Kit capable of quickly detecting amplification of incising incision enzyme nucleic acid of salmonella at constant temperature

Abstract

The invention relates to a primer and a probe capable of quickly detecting the amplification of incising incision enzyme nucleic acid of salmonella at a constant temperature. A positive primer sequence is SEQ ID NO:1; negative primer sequence is SEQ ID NO:2; a sequence of a P1 probe is SEQ ID NO:3; and the sequence of a P2 probe is SEQ ID NO:4. The invention also provides a kit and an applicationmethod of the same. The kit contains the sequences of the primer and the probe and is used for quickly detecting the salmonella through the amplification at the constant temperature. The primer and the probe of the kit are designed according to conservative gene sequences of bacterial strains to be detected, so that specificity of a detecting method is ensured. Moreover, the detecting method has a sensitivity similar to a PCR (Polymerase Chain Reaction) detecting method, and only an ordinary water bath kettle is needed, instead of an expensive PCR instrument. Results can be detected by a universal quick assay board which can be used for quickly detecting amplimers of nucleic acid, instead of being observed by adopting a gel electrophoresis method. The kit has the advantages of simple structure, quickness, safety and no pollution, and is particularly applicable to a food inspection organization.

Description

technical field

[0001] The invention belongs to the technical field of detection of pathogenic microorganisms, and in particular relates to a prescription kit for rapid detection of bacterial samples using a nicking endonuclease constant temperature amplification (NEMA) technology and an application thereof. Background technique

[0002] Salmonella (Salmonella spp) belongs to the enterobacteriaceae, including bacteria that can cause food poisoning, gastroenteritis, typhoid and paratyphoid, and is a class of zoonotic foodborne Gram-negative pathogenic bacteria. In addition to infecting humans, Salmonella can also infect many animals, including mammals, birds, reptiles, fish, amphibians and insects. Humans and animals can be asymptomatic carriers after infection, and can also show clinical symptoms. Salmonella may also increase morbidity or mortality, or reduce the reproductive productivity of animals. According to reports, among various bacterial food poisoning incidents in...

Examples

Embodiment 1

[0043] Embodiment 1: to the detection of Salmonella standard bacterial strain

[0044] Prepare Salmonella nicking endonuclease nucleic acid constant temperature amplification kit according to the following formula:

[0045] 1) Template pretreatment reaction solution:

[0046] Each 23μL contains 2.5μL 10×Taq Platinum buffer II, 1.0μL 2.5mmol / L dNTP, 1.0μL 10μmol / L forward primer, 1.0μL 10μmol / L reverse primer, 1.0μL 2.5U / μL Taq Platinum DNA Polymerase and 16.5μL wxya 2 O (sterilized double distilled water).

[0047] The forward primer SEQ ID NO: 1 described therein:

[0048] 5-CCTTCGCTCATTTTT GCTCTTC AATCGACGGACATCGACAGAC-3;

[0049] Reverse primer: SEQ ID NO: 2:

[0050] 5-AAGGGATCCGACAG GCTCTTC GCAGTACCTTCCTCAGCCTTG-3;

[0051] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0052] 2) NEMA constant temperature amplification reaction solution:

[0053] Each 46 μL contains 5 μL 10×NEBuffer 3, 1.0 μL 2.5 mm...

Embodiment 2

[0076] Example 2: Detection of Escherichia coli ATCC 25922

[0077] Prepare Salmonella nicking endonuclease nucleic acid constant temperature amplification kit according to the following formula:

[0078] 1) Template pretreatment reaction solution:

[0079] Each 23μL contains 2.5μL 10×Taq Plainum buffer II, 1.0μL 2.5mmol / L Dntp, 1.0μL 10μmol / L forward primer, 1.0μL 10μmol / L reverse primer, 1.0μL 2.5U / μL Taq Platinum DNA Polymerase and 16.5 μL ddH 2 O (sterilized double distilled water).

[0080] The forward primer SEQ ID NO: 1 described therein:

[0081] 5-CCTTCGCTCATTTTT GCTCTTC AATCGACGGACATCGACAGAC-3;

[0082] Reverse primer: SEQ ID NO: 2:

[0083] 5-AAGGGATCCGACAG GCTCTTC GCAGTACCTTCCTCAGCCTTG-3;

[0084] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0085] 2) NEMA constant temperature amplification reaction solution:

[0086] Each 46 μL contains 5 μL 10×NEBuffer 3, 1.0 μL 2.5 mmol / L dNTP, 1.0 μL ...

Embodiment 3

[0109] Example 3: Detection of food samples suspected of being infected with Salmonella

[0110] The food sample was subjected to DNA extraction and amplification detection according to the method described in Example 1, and the product was detected with a universal nucleic acid amplification rapid detection plate. Results The quality control area C showed a red band, and the detection area T also had a red band, indicating that the sample to be tested was infected by Salmonella.

[0111] The primers, probes and kits of the present invention can also detect Salmonella in other samples.

[0112]

[0113]

technical field

[0001] The invention belongs to the technical field of detection of pathogenic microorganisms, and in particular relates to a prescription kit for rapid detection of bacterial samples using a nicking endonuclease constant temperature amplification (NEMA) technology and an application thereof. Background technique

[0002] Salmonella (Salmonella spp) belongs to the enterobacteriaceae, including bacteria that can cause food poisoning, gastroenteritis, typhoid and paratyphoid, and is a class of zoonotic foodborne Gram-negative pathogenic bacteria. In addition to infecting humans, Salmonella can also infect many animals, including mammals, birds, reptiles, fish, amphibians and insects. Humans and animals can be asymptomatic carriers after infection, and can also show clinical symptoms. Salmonella may also increase morbidity or mortality, or reduce the reproductive productivity of animals. According to reports, among various bacterial food poisoning incidents in...

Examples

Embodiment 1

[0043] Embodiment 1: to the detection of Salmonella standard bacterial strain

[0044] Prepare Salmonella nicking endonuclease nucleic acid constant temperature amplification kit according to the following formula:

[0045] 1) Template pretreatment reaction solution:

[0046] Each 23μL contains 2.5μL 10×Taq Platinum buffer II, 1.0μL 2.5mmol / L dNTP, 1.0μL 10μmol / L forward primer, 1.0μL 10μmol / L reverse primer, 1.0μL 2.5U / μL Taq Platinum DNA Polymerase and 16.5μL wxya 2 O (sterilized double distilled water).

[0047] The forward primer SEQ ID NO: 1 described therein:

[0048] 5-CCTTCGCTCATTTTT GCTCTTC AATCGACGGACATCGACAGAC-3;

[0049] Reverse primer: SEQ ID NO: 2:

[0050] 5-AAGGGATCCGACAG GCTCTTC GCAGTACCTTCCTCAGCCTTG-3;

[0051] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0052] 2) NEMA constant temperature amplification reaction solution:

[0053] Each 46 μL contains 5 μL 10×NEBuffer 3, 1.0 μL 2.5 mm...

Embodiment 2

[0076] Example 2: Detection of Escherichia coli ATCC 25922

[0077] Prepare Salmonella nicking endonuclease nucleic acid constant temperature amplification kit according to the following formula:

[0078] 1) Template pretreatment reaction solution:

[0079] Each 23μL contains 2.5μL 10×Taq Plainum buffer II, 1.0μL 2.5mmol / L Dntp, 1.0μL 10μmol / L forward primer, 1.0μL 10μmol / L reverse primer, 1.0μL 2.5U / μL Taq Platinum DNA Polymerase and 16.5 μL ddH 2 O (sterilized double distilled water).

[0080] The forward primer SEQ ID NO: 1 described therein:

[0081] 5-CCTTCGCTCATTTTT GCTCTTC AATCGACGGACATCGACAGAC-3;

[0082] Reverse primer: SEQ ID NO: 2:

[0083] 5-AAGGGATCCGACAG GCTCTTC GCAGTACCTTCCTCAGCCTTG-3;

[0084] The underlined part in the forward primer and the reverse primer is the nicking endonuclease recognition site;

[0085] 2) NEMA constant temperature amplification reaction solution:

[0086] Each 46 μL contains 5 μL 10×NEBuffer 3, 1.0 μL 2.5 mmol / L dNTP, 1.0 μL ...

Embodiment 3

[0109] Example 3: Detection of food samples suspected of being infected with Salmonella

[0110] The food sample was subjected to DNA extraction and amplification detection according to the method described in Example 1, and the product was detected with a universal nucleic acid amplification rapid detection plate. Results The quality control area C showed a red band, and the detection area T also had a red band, indicating that the sample to be tested was infected by Salmonella.

[0111] The primers, probes and kits of the present invention can also detect Salmonella in other samples.

[0112]

[0113]

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