Establishment Of A Nucleic Acid Detection Technique For Common Bacteria In Bloodstream Infection Based On RPA

Objective Bloodstream infection is a systemic infectious disease in which various pathogenic microorganisms invade the human blood and reproduce rapidly in the blood,leading to multiple organ dysfunction of the body and shock or even death in severe cases.If the infected pathogen is not identified in time,the use of empirical broad-spectrum antibiotics and ineffective antibiotics may lead to more serious consequences,while the traditional blood culture currently used in clinical laboratories take a long time,increase the financial burden on patients and reduce the survival rates.Molecular diagnostic methods such as mass spectrometry and traditional PCR have high professional and technical requirements for personnel,and the equipment is expensive,which is not suitable for field testing outside primary hospitals and laboratories,which is not conducive to the early detection of patients with bloodstream infection.Isothermal amplifification techniques have an irreplaceable advantage,that does not require complex equipment.Bloodstream infection caused by bacteria is the most common.As a result,The rapid detection technique of common bacteria,including Staphylococcus aureus,Escherichia coli,Klebsiella pneumoniae,based on RPA is established and optimized,aiming to establish a method to quickly detect common bacteria in bloodstream,shorten the treatment time and improve the prognosis.Methods 1.According to the national bloodstream infection statistics,the top three bloodtream infection bacteria,Staphylococcus aureus,Escherichia coli,Klebsiella pneumoniae that is target bacteria and other common bacteria,Pseudomonas aeruginosa,Enterobacter cloacae,Staphylococcus epidermidis,enterococcus faecium,enterococcus faecalis that verify system specificity,were extracted DNA.2.Establishment and optimization of detection technique based on RPA:According to highly conserved sequences of Staphylococcus aureus,Escherichia coli and Klebsiella pneumoniae,9pairs of specific primers were designed to establish and optimize the amplification system.3.Detection technique compared with PCR was evaluated from three aspects of sensitivity,specificity and repeatability.4.Clinical blood culture samples were collected and detected by RPA and PCR,that was evaluated the clinical value.Results The detection system based on RPA showed good repeatability,specificity and sensitivity.The limit of detection technique for Staphylococcus aureus based on RPA was 1×10~1copies/μl,that was 1000 times than PCR.7 blood culture specimens were detected,whose results showed 6 positive specimens and 1 negative specimen by RPA,that was consistent with the mass spectrometry results.While the blood culture specimens were detected by PCR,whose results showed 5 positive specimens and 2negative specimen.There is one false negative specimen compared with the result of mass spectrometry.The limit of detection technique for Escherichia coli based on RPA was 1×10~1copies/μl,that was 100 times than PCR.10 blood culture specimens were detected,whose results showed 9 positive specimens and 1 negative specimen by RPA,that was consistent with the mass spectrometry results.While the blood culture specimens were detected by PCR,whose results showed 7 positive specimens and 3negative specimen,There are 2 false negative specimens compared with the result of mass spectrometry.The limit of detection technique for Klebsiella pneumoniae,based on RPA was 1×10~3copies/μl,which was 100 times than PCR.13 blood culture specimens were detected,whose results showed 11 positive specimens and 2 negative specimen by RPA,which was consistent with the mass spectrometry results.While the blood culture specimens were detected by PCR,whose results showed 5 positive specimens and 8negative specimen,There are 6 false negative specimens compared with the result of mass spectrometry.Conclusion The established detection system of common bacteria in bloodstream based on RPA,which has a good application prospect,is rapid,specific,accurate and stable,and the equipment required is simple,which is conducive to detect patients with bloodstream infection early,improve the survival rate of patients,reduce the economic pressure of patients.