RPA-LbCas12a system-based composition for visual detection of nasal-like carbuncle and application of RPA-LbCas12a system-based composition

Abstract

The invention relates to the technical field of molecular diagnosis, in particular to a composition for visually detecting nasosphallex-like carbuncle based on an RPA-LbCas12a system and application of the composition. The invention provides a composition for detecting Burkholderia pseudomallei. The composition comprises an RPA (recombinase polymerase amplification) primer, crRNA (complementary Ribonucleic Acid), LbCas12a protease and an ssDNA (single-stranded deoxyribonucleic acid) fluorescent probe, the nucleotide sequences of the RPA primer are as shown in SEQ ID NO: 5 and SEQ ID NO: 6; the nucleotide sequences of the transcription template of the crRNA are shown as SEQ ID NO: 7 and SEQ ID NO: 9. Based on the composition, the invention also provides a method for detecting the burkholderia pseudomallei. According to the method, through combination of RPA amplification and a CRISPR-Cas12a system, the sensitivity with the lower detection limit of 2.7 copies / mu L and higher specificity are obtained, whether a sample contains the burkholderia pseudomallei or not can be detected within 1 h, large instruments and equipment are not needed, and rapid diagnosis and screening of the burkholderia pseudomallei are facilitated.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to a composition for visual detection of Burkholderia pseudomallei based on an RPA-LbCas12a system and an application thereof. Background technique [0002] Melioidosis (Melioidosis) is a disease caused by Burkholderia pseudomallei (Bp) infection through respiratory aerosols or skin ulceration, and its natural foci are subtropical areas such as Hainan and the southeast coast of my country. . With the increase in population mobility in recent years, imported cases have also occurred in unnatural foci. At present, there is no effective treatment plan for BP infection, and there is no vaccine protection; Bp is easy to obtain, easy to spread, and widely resistant to resistance, and has been included in the international "Biological Weapons Convention" verification list and the US CDC Class I bioterrorism agent; the clinical manifestations of infection are "occult" Or "like ...

AI Technical Summary

Problems solved by technology

Although isolation and culture is the gold standard for infection diagnosis, it takes a long time, and it is easy to miss the best opportunity for diagnosis and treatment. In addition, if there is no relevant experience, it is easy to be misidentified as pollution or other bacteria of the same species, which will lead to wrong treatment and delay the patient's condition; Due to the interference of the window period and the background of the population in the epidemic area (the positive rate of serum antibodies in the local population in Hainan is 6% to 20%), it is very easy to cause false negative or false positive results in serological detection; Bacterial mass spectrometry identification based on proteomics analysis has no effect on bacteria. The concentration and purity of the sample are required, and the detection results of some strains are unstable due to regional differences and database update lags; currently reported molecular biology methods are mainly based on PCR methods and Lamp (loop-mediated Isothermal amplification), detection of target genes including 16s rRNA and type III and type VI secretion system gene clusters, etc., 16s rRNA of Burkholderia pseudomallei and Burkholderia thailand and Burkholderia The sequence difference is small, which can easily lead to identification errors; the PCR method has high requirements on instrument conditions and personnel operations, and is not suitable for bedside rapid diagnosis (quick detection in the field); and the Lamp method is easy to form aerosol pollution, resulting in false positives result

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