A detection kit for Salmonella typhi, its preparation method and its application

Abstract

The invention discloses a detection kit for Salmonella typhi, its preparation method and application thereof, and the detection kit includes: amplification primers, crRNA, LbCas12a-RR protein and a single-stranded DNA reporting system; crRNA is for the flag gene of Salmonella typhi The specific crRNA of the detection segment; the sequence expressed by the LbCas12a-RR protein is the prokaryotic codon optimized with the Cas12 protein nucleic acid sequence, and the 532 and 595 amino acids are mutated to the sequence after G532R and K595R respectively; the application of the kit, The detection kit for Salmonella typhi is used for nucleic acid detection of Salmonella typhi; the kit has high sensitivity, strong specificity, and rapid visualization.

Description

technical field

[0001] The invention relates to a detection kit for Salmonella typhi, a preparation method and application thereof, and belongs to the technical field of microbial detection. Background technique

[0002] Salmonella enterica serotype typhoid (S. Typhi) is a human host-restricted pathogen, and typhoid fever caused by it remains a major public health problem. It is estimated that about 10.9 million people around the world are infected with typhoid every year, and nearly 100,000 people die from it, with children having the highest infection rate. Salmonella typhi is mainly spread through water or food contaminated with feces. When a patient ingests contaminated food or water, Salmonella typhi will invade the intestinal mucosa and spread to the whole body, causing acute symptoms such as high fever, hepatosplenomegaly, and rash. Since the symptoms and signs caused by Salmonella typhi are non-specific, it is difficult to diagnose directly through clinical symptom...

Examples

preparation example Construction

[0051] The preparation method of the detection kit of above-mentioned Salmonella typhi comprises:

[0052] Amplification primer preparation step: designing amplification primers containing crRNA, the length of the amplification primers is 30-37bp;

[0053] crRNA preparation step: aiming at the Salmonella typhi flag gene (sequence SEQ ID NO.5), looking for targeting sequences comprising LbCas12a-RR recognition sequence (PAM) TTTN and TCTN, designing and preparing crRNA with a length of 23bp;

[0054] LbCas12a-RR protein preparation steps: Carry out prokaryotic codon optimization for the Cas12 protein nucleic acid sequence and mutate amino acids at positions 532 and 595 to G532R and K595R, respectively, to obtain the sequence expressed by the LbCas12a-RR protein, as shown in SEQ ID NO.6, After the LbCas12a-RR protein was transformed, the recognition region was expanded, and it could recognize both TTTN and TCTN; then it was constructed into the pET28a expression vector, and the ...

Embodiment 1

[0060] Nucleic acid preparation of Salmonella typhi:

[0061] For the flag gene fragment of Salmonella typhi, the amplification primers were designed according to the sequence of NCBI CMCC50071 strain, synthesized by Nanjing GenScript Company, named flag-PCR-F and flag-PCR-R, and the corresponding sequences are SEQ ID NO.7 and SEQ ID NO.7, respectively ID NO.8.

[0062] A DNA nucleic acid sample corresponding to the flag gene of Salmonella typhi was prepared by PCR. The specific operation is as follows: the standard strain of Salmonella typhi (CMCC50071) was obtained from the Center for Disease Control and Prevention, the strain was recovered and amplified, and the bacterial genomic DNA was extracted using the bacterial genomic DNA extraction kit (TIANGEN). Max Super-FidelityDNA Polymerase (Vazyme P505), using Salmonella typhi genomic DNA as a template to amplify the DNA sample pcS.Typhi-flag corresponding to the flag gene. Purify with MEGAclear kit (Thermo Fisher Scientifi...

Embodiment 2

[0064] Kit crRNA preparation and detection:

[0065] According to the targeting sequences of specific PAM TTTN and TCTN recognized by LbCas12a-RR, 20 specific crRNAs were designed on the target sequence of the flag gene, and the sequences are shown in Table 1:

[0066] Design sequence of table 1crRNA

[0067] serial number crRNA nomenclature sequence SEQ ID NO.3 flag-crRNA1 TCTGCGAATGGTACTAACTCCCAGTCT SEQ ID NO.9 flag-crRNA2 TCTTTTAAATCAATATCGATAGTTTCA SEQ ID NO.4 flag-crRNA3 TTTAAAAGAAATCAGCTCTAAAACACT SEQ ID NO.10 flag-crRNA4 TTTTAGAGCTGATTTCTTTTTAAATCAA SEQ ID NO.11 flag-crRNA5 TTTGTCTTCCGGTCTGCGTATCAAC SEQ ID NO.12 flag-crRNA6 TTTTACCGCGAACATCAAAGGTCTGAC SEQ ID NO.13 flag-crRNA7 TTTCACGCCGTTGAACTGAGTCTGGCC SEQ ID NO.14 flag-crRNA8 TTTTAGCAGTAATTGCACCTGTTTTCT SEQ ID NO.15 flag-crRNA9 TTTGAGCAACGCCAGTACCATCTGTAT SEQ ID NO.16 flag-crRNA10 TCTGCGCAATGGAGATACCGTCGTTAG SEQ...

technical field

[0001] The invention relates to a detection kit for Salmonella typhi, a preparation method and application thereof, and belongs to the technical field of microbial detection. Background technique

[0002] Salmonella enterica serotype typhoid (S. Typhi) is a human host-restricted pathogen, and typhoid fever caused by it remains a major public health problem. It is estimated that about 10.9 million people around the world are infected with typhoid every year, and nearly 100,000 people die from it, with children having the highest infection rate. Salmonella typhi is mainly spread through water or food contaminated with feces. When a patient ingests contaminated food or water, Salmonella typhi will invade the intestinal mucosa and spread to the whole body, causing acute symptoms such as high fever, hepatosplenomegaly, and rash. Since the symptoms and signs caused by Salmonella typhi are non-specific, it is difficult to diagnose directly through clinical symptom...

Examples

preparation example Construction

[0051] The preparation method of the detection kit of above-mentioned Salmonella typhi comprises:

[0052] Amplification primer preparation step: designing amplification primers containing crRNA, the length of the amplification primers is 30-37bp;

[0053] crRNA preparation step: aiming at the Salmonella typhi flag gene (sequence SEQ ID NO.5), looking for targeting sequences comprising LbCas12a-RR recognition sequence (PAM) TTTN and TCTN, designing and preparing crRNA with a length of 23bp;

[0054] LbCas12a-RR protein preparation steps: Carry out prokaryotic codon optimization for the Cas12 protein nucleic acid sequence and mutate amino acids at positions 532 and 595 to G532R and K595R, respectively, to obtain the sequence expressed by the LbCas12a-RR protein, as shown in SEQ ID NO.6, After the LbCas12a-RR protein was transformed, the recognition region was expanded, and it could recognize both TTTN and TCTN; then it was constructed into the pET28a expression vector, and the ...

Embodiment 1

[0060] Nucleic acid preparation of Salmonella typhi:

[0061] For the flag gene fragment of Salmonella typhi, the amplification primers were designed according to the sequence of NCBI CMCC50071 strain, synthesized by Nanjing GenScript Company, named flag-PCR-F and flag-PCR-R, and the corresponding sequences are SEQ ID NO.7 and SEQ ID NO.7, respectively ID NO.8.

[0062] A DNA nucleic acid sample corresponding to the flag gene of Salmonella typhi was prepared by PCR. The specific operation is as follows: the standard strain of Salmonella typhi (CMCC50071) was obtained from the Center for Disease Control and Prevention, the strain was recovered and amplified, and the bacterial genomic DNA was extracted using the bacterial genomic DNA extraction kit (TIANGEN). Max Super-FidelityDNA Polymerase (Vazyme P505), using Salmonella typhi genomic DNA as a template to amplify the DNA sample pcS.Typhi-flag corresponding to the flag gene. Purify with MEGAclear kit (Thermo Fisher Scientifi...

Embodiment 2

[0064] Kit crRNA preparation and detection:

[0065] According to the targeting sequences of specific PAM TTTN and TCTN recognized by LbCas12a-RR, 20 specific crRNAs were designed on the target sequence of the flag gene, and the sequences are shown in Table 1:

[0066] Design sequence of table 1crRNA

[0067] serial number crRNA nomenclature sequence SEQ ID NO.3 flag-crRNA1 TCTGCGAATGGTACTAACTCCCAGTCT SEQ ID NO.9 flag-crRNA2 TCTTTTAAATCAATATCGATAGTTTCA SEQ ID NO.4 flag-crRNA3 TTTAAAAGAAATCAGCTCTAAAACACT SEQ ID NO.10 flag-crRNA4 TTTTAGAGCTGATTTCTTTTTAAATCAA SEQ ID NO.11 flag-crRNA5 TTTGTCTTCCGGTCTGCGTATCAAC SEQ ID NO.12 flag-crRNA6 TTTTACCGCGAACATCAAAGGTCTGAC SEQ ID NO.13 flag-crRNA7 TTTCACGCCGTTGAACTGAGTCTGGCC SEQ ID NO.14 flag-crRNA8 TTTTAGCAGTAATTGCACCTGTTTTCT SEQ ID NO.15 flag-crRNA9 TTTGAGCAACGCCAGTACCATCTGTAT SEQ ID NO.16 flag-crRNA10 TCTGCGCAATGGAGATACCGTCGTTAG SEQ...

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