Development And Evaluation Of A Nucleic Acid Testing Assay For Detection Of Bacterial Contamination In Platelets

Recently,bacterial contamination in platelet concentrates(PCs)is becoming a worldwide concerned problem as the risks of transfusion-related viral infectious diseases have decreased to a low level and the platelet concentrates(PCs)have been widely uesd in clinic.According to the literature,the incidence of bacterial contamination is still approximately 1:3,000 in single donor plateletpheresis(SDP)and 1:2,000 in whole-blood random donor platelets(RDP).This risk is estimated to be 50- to 250-fold higher than the combined risk of transfusion-related infections associated with hepatitis B Virus(HBV),hepatitis C Virus(HCV),human immunodeficiency virus type 1(HIV-1)and human T-cell leukemia virus typesⅠandⅡ(HTLV-Ⅰ/Ⅱ).So the sepsis caused by bacterial contamination is considered as the most common and serious risk.At present,the US-FDA has cleared the BacT/ALERT,Pall eBDS as well as Scansystem for bacterial contamination testing of platelet products to reduce the risk transfusion-associated septic reacting.And the BacT/ALERT 3D automated culturing syste are used in many blood banks of developed country for routine bacterial screening.It is sensitive,reliable even easy handling.But there may still got some obstacles couldn't be resolved such as the long detecting time and cost on the bottles. And the research on this field is relatively poor in our country.A real-time fluorescence quantitative PCR assay was developed and validated to detect bacterial DNA in PLTs.With this universal real-time PCR,analytically it is possible to detect a single copy of bacterial DNA within a few hours.However,as lack of an efficient way to extract bacterial DNA and a measure to decrease the quantity of contaminated DNA in real-time PCR,this assay couldn't be used in clinic for routine bacterial screening.So our research was studied on the bacterial DNA extraction at the beginning. The gram-positive bacterium(Staphylococcus aureus)was chosen for this study.After comparing the five classical bacterial DNA extraction ways we found that the highest efficient method is Chelex-100.Then some improvements were taken to ensure the sensitivity of this method which is 0.2 CFU equivalents per PCR(corresponds to 10CFUs/ml)at the low copies of bacterial ribosomal DNA.In our study,real-time PCR amplification was performed with a set of universal primers and probes targeting eubacterial 16S ribosomal DNA.The Microcon YM-100 Centrifugal Filter Units and Mltrafree-MC double-filtration technique was used to decrease potential sources of contaminating bacterial DNA and improve the sensitivity and specificity of the assay.The background of the negative control was decreased dramatically after the PCR mixture was double-filtrated.The threshold cycles(Ct)values of the low copies of bacterial ribosomal DNA(0.2 to 0.3 CFU equivalents per PCR)were before 30,while the Ct value of the negative control was significantly delayed to 34.There was significant different(p＜0.001)between them. So the double-filtration technique could decrease the background signal and improve the sensitivity and specificity of the real-time PCR assay.The practicability of this 16s ribosomal DNA real-time PCR assay for detection of bacterial contamination in platelet concentrate was also evalued.The results showed that the detection limit of the PCR method was 0.2-0.3 CFU equivalents per PCR in all six transfusion-associated bacteria,which had a significant different(p＜0.001)from negative controls.Comparing with the BacT/ALERT 3D automated culturing system,real-time PCR in combination with the modified chelex-100 DNA extraction method we developed was very reliable.The sensitivity and specificity were 100%respectively.And the kappa was 1.000A total of 447 spiked PCs and 470 sterile PCs were tested by the real-time PCR assay,and the data were analyzed by the Receiver Operating Characteristic(ROC) Curve.ROC curve analysis on the Ct value of the clinical samples showed that the area under the ROC Curve was 1.000,which means this PCR assay had a very good diagnosis value.Many bacteria may develop as L-forms when bacteria are exposed to compounds which inhibit cell wall synthesis.These L-forms are wallless but keep the pathogenic. There are many chronic infection related with L-forms.Meanwhile L-forms can't be detected by ordinary medium.This must be threatening to the safty of blood transfusion.Our reaserch using disk diffusion method for drug sensitivity test induced S.aureus to L-forms.Then L-forms were detected by the Real-Time PCR methods in a few hours.So the Real-Time PCR method for detection of L-forms is convenient,less time, which ensured the safety of blood products.The Real-time PCR has a lot of advantages on manipulation,sensitivity as well as specificity,which is more suitable for the screening of PCs just before release. Although our research was only in laboratory,and lots of work have to be done before which implemented on clinic.But this 16s rDNA gene Real-time PCR could be wildly used in the future for detection of bacterial contamination in platelets.