Detection Of Pathogenic Microorganisms After Organ Transplantation Based On Second Generation Sequencing Technology Platform

Solid organ transplantation(SOT)is an effective method for treating a variety of end-stage organ diseases.Postoperative rejection and infection and other complications are the main factors that affect the survival time of patients.Transplant recipients are required to take immunosuppressants for a long time after surgery to reduce the risk of postoperative rejection.However,due to the use of immunosuppressants,the risk of postoperative infection of patients is relatively increased,which threatens the survival of patients after surgery.At present,clinical infection diagnosis depends on the doctor’s initial diagnosis of the patient’s clinical signs,and then collecting clinical specimens for further testing.There is no uniform gold standard for the detection of multiple pathogenic microorganisms in clinical practice,and the defects of routine clinical testing or long detection period,weak specificity,or low sensitivity,etc.,which cannot quickly and effectively detect pathogenic microorganisms after organ transplantation.Next-generation sequencing(NGS)technology has high throughput,short cycle,high sensitivity and strong specificity,which can simultaneously detect various pathogenic microorganisms.For immunosuppression after organ transplantation,patients with weak clinical signs and delayed The detection of pathogenic microorganisms has a very broad application prospect.Therefore,this paper establishes a method of applying the next generation sequencing technology platform to detect pathogenic microorganisms in infected patients after organ transplantation,which can detect and validate the main pathogenic microorganisms after organ transplantation.A nested PCR detection method for BKV DNA was established to investigate the distribution of BKV DNA in peripheral blood of kidney transplant patients.(1)Through the use of WGS and hybrid capture sequencing technology,474 samples from 136 organ transplant patients collected from six domestic organ transplant centers in 2017-2019 were tested.All samples have successfully detected pathogenic microbial DNA,and the detection specificity is strong,which can beaccurate to specific types and strains.The detection sensitivity is high,and at least one pathogenic microbial DNA can be detected.(2)According to the clinical prompts related to the risk of infection after organ transplantation of the collected samples(increased creatinine,fever,or inflammatory response,etc.),patients were divided into prompted and non-prompted groups,and the sequencing results of the two groups of patient samples were analyzed.The results showed that the copy number of pathogenic microorganisms in the clinical prompted group was significantly higher than that in the non-prompted group,which was consistent with the clinical preliminary diagnosis results.In addition,copies of pathogenic microorganisms were also detected in the samples of the non-prompted group.The reason for this result may be that some patients have latent pathogenic microorganisms in the body,but no large-scale replication has occurred and infection has not been caused.This result indicates that the next-generation sequencing technology can not only assist clinicians in diagnosis of infection,but also indicate the risk of infection by potential pathogenic microorganisms.(3)After analysis,we screened eight major pathogenic microorganisms after organ transplantation(BKV,EBV,CMV,Pseudomonas Aeruginosa,Acinetobacter Baumannii,Staphylococcus Aureus,Klebsiella Pneumoniae,and Pneumocystis Carinii).We studied the detection effect of the above pathogenic microorganisms in the included samples.Through multiple PCR and then sequencing,the above pathogenic microorganisms were successfully detected in the sample,and it was verified that the designed primer has strong specificity and high sensitivity.In addition,multiple PCR combined with next-generation sequencing has a short experimental period and low cost.This method is expected to be widely used in the detection of pathogenic microorganism DNA and even functional genes such as drug resistance genes and virulence genes in clinical patient samples.(4)Nested PCR was used to investigate the distribution of BKV DNA in peripheral blood of kidney transplant patients.The results showed that when BKV viremia occurred after surgery,BKV replicated at high levels in peripheral blood.Along with the escalation of the infection level,when BKVN is complicated,BKV in the patient’s peripheral blood gradually infects the patient’s peripheral blood leukocytes.As the disease progresses from BKVN stage A to stage B and even C,the BKV load in leukocytes further increases.Which causes serious related diseases,BKV is difficult to clear from the patient at this time.In summary,the next-generation sequencing technology has broad application prospects in the detection of pathogenic microorganisms after organ transplantation and even the detection of infection in routine clinical patients.This technology can effectively detect the pathogenic microorganisms caused,and even the pathogenic microorganisms that indicate the potential risk of infection.This technology can effectively help clinicians to monitor the flora of patients and adjust the treatment plan in time,which is beneficial to the long-term survival of patients.