| Cronobacter sakazakii(C.sakazakii)and Salmonella are two common sources of contamination in powdered infant formula(PIF)with high morbidity,leading to rare but life-threatening neonatal meningitis,sepsis and necrotizing enterocolitis.There are many cases of illness or even death of newborns due to the consumption of the contaminated PIF.Therefore,the establishment of rapid and effective detection methods is conducive to the prevention of disease and better escort for food safety.Traditional microbiological detection methods,such as medium culture,biochemicalserum identification,and morphological characteristics observation,etc.,are cumbersome,time-consuming,and unable to rapidly quantify harmful targets.The purpose of this study is to establish a real-time quantitative recombinase polymerase amplification(qRPA)technique for the simultaneous and rapid detection of C.sakazakii and Salmonella in PIF.It aims to explore and study the multiplex detection and identification of live bacteria,and to evaluate the detection ability of this technology in food samples by artificial contamination.The main research contents and results are as follows:1.Optimization and establishment of multiplex qRPA methodA specific probe was designed for outer membrane protein A(omp A)of C.sakazakii,different primers were designed around the localization of the probe,and then cross-matched to determine the best combination of sensitivity/specificity.For the invasion protein A(inv A)of Salmonella,the same method as that of C.sakazakii was used to determine the best set of primers for sensitivity/specificity.By optimizing the concentrations of primers and probes,the two optimal combinations were constructed a multiplex system of amplification conditions to simultaneously detect two target pathogenic bacteria.The results showed that:(1)The reaction temperature of this method was 37°C,the reaction time was 20 min;(2)The concentration of probe and primers of C.sakazakii was 200 n M and 60 n M,and the concentration of probe and primers of Salmonella was 400 n M and 120 n M;(3)The detection limit of C.sakazakii was 10~4cfu/m L and the detection limit of Salmonella was 10~3cfu/m L.2.Application of multiplex qRPA method in artificially spiked PIFIn order to evaluate the detection ability of multiplex qRPA method in the actual infant formula samples,10-fold series dilutions of C.sakazakii and Salmonella were respectively spiked into the sterile PIF as samples to be tested by artificial contamination method.The results showed that the detection limit of this method in PIF was consistent with that in pure culture,10~4cfu/g and 10~3cfu/g for C.sakazakii and Salmonella respectively.Considering the low level of contamination in the food samples,the detection of the target number of bacteria below the detection limit level after the pre-enrichment step was evaluated.The results showed that Salmonella with an inoculation level of 0.1 cfu/g could be detected after 8 h of pre-enrichment at37°C;the C.sakazakii with an inoculation level of 0.1 cfu/g could be detected after 10 h of pre-enrichment at 37°C.3.Detection of viable C.sakazakii in PIF using nucleic acid intercalator combined with q PCR and qRPAThis study successfully combined the improved propidium monoazide(PMAxx)with qRPA to quantitatively analyze the viable C.sakazakii in PIF.Specific primers and probes were designed for the alpha-glucosidase(glu A)gene sequence of C.sakazakii,heat-treated inactivation method was used to prepare dead bacteria by heating at 90°C for 15 min.The effects of different concentrations of PMAxx and different amplicon lengths on the dead bacteria were tested.The results showed that the amplicon length and PMAxx concentration hadsignificant effects on dead cells.PMAxx at the optimal concentration of 10?g/m L combined with a 256 bp qRPA amplification length could completely achieve suppression of 10~8cfu/m L of dead cells,while q PCR required a longer amplification fragment(338bp).In the application of spiking PIF,PMAxx-qRPA could detect 10~3cfu/g of viable C.sakazakii,and the detection limit of PMAxx-q PCR was 10~2cfu/g.In the PMAxx-q PCR method,the matrix affected the treatment effect of PMAxx,but in the PMAxx-qRPA method,this phenomenon did not occur.After mixing 0,10~7,10~6and 10~5cfu/g viable cells with 10~7cfu/g dead cells in the PIF matrix,PMAxx-qRPA and PMAxx-q PCR methods completely eliminate false positive signals of dead cells(false positive rate was 0).By comparing with PMAxx-qRPA,PMAxx-q PCR,q PCR,qRPA and plate counting for quantifying C.sakazakii in PIF,both PMAxx-qRPA and PMAxx-q PCR were obviously superior to q PCR and qRPA,with consistency to plate counting.In conclusion,these two methods established had their own advantages.Among them,PMAxx-q PCR had higher sensitivity and PMAxx-qRPA had a faster detection speed.Both of them could be used as effective tools to quantify viable C.sakazakii in PIF. |
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