| 1. Background and objectiveCronobacter spp. is an important new type of foodborne pathogen, which includes all Enterobacter sakazakii of the previous taxonomy system. This genus of bacteria resides in the intestinal tract of human and animals, is peritrichous, motile, facultative anaerobic, gram-negative and sporeless. This bacteria is distributed worldwide, and infects the digestive tract of children primarily through formula milk powder, resulting in neonatal meningitis, sepsis, and necrotizing enterocolitis, or even neurological sequelae and death.Since the first two cases of Cronobacter spp. infection-caused meningitis in1961, Cronobacter spp. infection-related diseases have been reported worldwide. In April2001, at a hospital in the US, a premature infant was sent to neonatal intensive care unit because of fever and tachycardia. Cronobacter spp. was found in the cerebrospinal fluid, and it was diagnosed as meningitis. Antibiotics failed to control the infection, and the baby died9days later. Expanded inspection was carried out for the49infants using feces and urine as samples, and10of them were found to be Cronobacter spp.-positive, which was believed to be from a batch of Portagen infant formula milk powder. Cronobacter spp. was actually detected in that batch of milk powder, which lead to the recall of Portagen infant formula milk powder in April2002. In1998,12infants that were fed with the brand of infant formula milk powder developed necrotizing enterocolitis, and Cronobacter spp. was isolated in feces of all these infants and that batch of milk powder. The2004Anhui Fuyang inferior infant formula milk powder incident aroused great attention of our government, and11strains of Cronobacter spp. were isolated from87samples of infant formula milk powder, the detection rate was up to12.16%. Although experts have declared that it was disproportional nutrition of the milk powder that caused deformities and death of the infants, the manifestations of these infants, including head enlargement and unresponsiveness indicated Cronobacter spp. infection, which might have played an important part in the disease. Cronobacter spp. is widely distributed, not only in contaminated food and food factories, it can actually be isolated from almost all places of the environment. Cronobacter spp. have been separated so far from chees, pork, vegetables, grains, herbs, spices, UHT-sterilized milk and other foods. Cronobacter spp. infection has a fatality rate of10-80%, seriously threating public health and safety, and as a conditional pathogen, it is most dangerous for infants.Currently, methods to detect Cronobacter spp. include isolation and culture (recommended by FDA), biochemical and serological identification, immunological methods and PCR. Traditional methods to detect and quantify Cronobacter spp. use selective medium, and involve enrichment of bacteria and selectively culture of target bacteria. This method has three major shortcomings:first, the pathogenic bacteria generally exists in trace amount, so it can be easily missed during sample handling and detection; second, it’s remarkably tedious and time-consuming, the detection process generally requires over6days; and third, many pathological bacterial are difficult to culture or nonculturable, so this method has low sensitivity. As for the other methods, immunological methods has low specificity and sensitivity; PCR is sensitive, accurate and fast, and can replace traditional detection method, but it requires expensive instrument, tedious electrophoresis process, and professional technique of the personnel, which make it difficult to promote and popularize this method in grassroots units. Therefore, it is of great importance for epidemiology study and prevention of Cronobacter spp. infection to establish a highly specific, rapid, simple and accurate detection method.1) Sample enrichment by immunomagnetic separation (IMS)Biological samples are often complex mixtures, which can’t be directly used for detection and requires preparation except for a few special samples. Traditional sample processing aims to isolate the target microorganism by selective culture to obtain purified microorganism. If damaged or thermal-stressed, the microorganism would need extra enrichment culture, including pre-enrichment, selective enrichment, and post-enrichment, so it would cost a lot of time to detect specific microorganism from the samples. In practice, most of the time is spent on enrichment of the microorganism, which makes rapid detection virtually impossible. Immunomagnetic separation can specifically absorb and enrich target bacteria, and has attracted much attention of the scholars.Immunomagnetic beads are3-5μm, paramagnetic beads with good dispersion and coated with specific monoclonal antibody. Immunomagnetic separation was developed since the mid-1970s, and the separation principle is that the coating antibody binds specific marker on the surface of target cells, so that antigen-positive and antigen-negative cells can be separated. This method combines the intrinsic advantages of solidified agent and the high specificity of immuno reaction, is widely applied in separation and purification of multiple proteins, and is well suited for isolation and detection of pathogenic microorganisms in food, water, biological samples, and environmental samples, showing good perspective of development and application.The principle of application of IMS in detection of microorganism:magnetic beads are coated with specific antibody that captures target microorganism in the sample or enriched culture, then the beads are attracted and precipitated with magnetic field. So target bacteria can be isolated from interfering bacteria and sample residue, getting concentrated, so that detection sensitivity and detection rate can be improved. IMS can be easily operated, the immunomagnetic beads are added to sample or enriched culture, incubated at room temperature for a certain period of time, then washed with butter several times in magnetic field, and the target bacteria is captured and concentrated, which can be directly used for subsequent experiments without extra separation from the beads. This method has the advantages of rapid separation, high efficiency, good repeatability, simple operation without expensive instruments, and little effects on the biological characteristics and function of the target cell or biological substances, and is thus extremely suited for detection of microorganism.2) Rapid detection by loop-mediated isothermal amplification (LAMP)Loop-mediated isothermal amplification is a novel isothermal nucleic acid amplification technology developed by Japanese scholar Notomi and his colleagues. Briefly, the template is incubated with4or6primers specific for6or8region of target gene and a special strand displacement DNA polymerase (Bst DNA polymerase) under isothermal condition (about65℃), and the amplification reaction is completed. LAMP can amplify target DNA rapidly with high efficiency, high sensitivity and high specificity, in addition, this method can be easily operated and cost-efficient, thus is well suited for large-scale analysis and grassroots units, and it is now widely used in diagnosis of disease, detection of pathogenic microorganism, and sex identification of animal embryo.So, in this study, we combined the two techniques, that is, Cronobacter spp. is isolated and concentrated from sample, then used for rapid LAMP detection. First, the technique using IMS to capture Cronobacter spp. was established and tested with simulated sample. Then internal and external primers specific for the OmpA gene encoding a specific outer membrane protein of Cronobacter spp., and LAMP technique was used to detect Cronobacter spp. from the enriched sample, thus establishing the rapid and highly sensitive IMS-LAMP method for detection of Cronobacter spp. Then we tested this method with simulated dairy product sample and209samples of formula milk powder in market, and the results were compared with that of traditional national standard method.2. Method:1) The technique using IMS to capture Cronobacter spp. was established and tested using simulated sample. Bacterial suspension of Cronobacter spp. and other bacteria(escherichia coli, Vibrio parahaemolyticus, Shigella flexneri, Proteus vulgaris, salmonella typhimurium, staphylococcus aureus, etl) were prepared, and were captured using Cronobacter spp.-specific immunomagnetic beads, then the captured bacteria were seeded on plates for colony count, to calculate the recovery rate of IMS capturing. Meanwhile, interference assay was performed, in which different concentrations of Cronobacter spp. were added to E. coli suspension with a concentration of104cfu/ml, and then captured with IMS to assess its efficiency of specific enrichment. Simulated milk powder sample containing known amount of target bacteria were tested for IMS capturing after3h or6h of pre-enrichment and simulated feces sample was also tested without pre-enrichment, to evaluate the practical applicability of IMS-based method in detection of Cronobacter spp.2) Primers specific for a conserved region of the OmpA gene (Accession number:DQ000206) encoding an outer membrane protein of Cronobacter spp. were designed for LAMP. The parameters of amplification were optimized and a25μL LAMP reaction system was established.3) Genomic DNA of20strains of experimental bacteria were used as template for LAMP and PCR analyses, the specificities of the two were compared; Cronobacter spp. cultured overnight was diluted to density gradient and used for amplification by LAMP and PCR, to compare sensitivities of the two methods.4) Test using simulated sample:three kinds of Cronobacter spp.-negative milk powder were inoculated with ATCC29544Cronobacter spp. strain, and were used for detection by LAMP and PCR, and the results were compared with that of national standard method.5)209samples of domestic infant formula milk powder were collected, and tested using the IMS-LAMP technique, and the results were compared with that of national standard.3. Results1) Colony count showed that recovery rates of IMS for the ATCC29544, ATCC51329and lab isolated strains of Cronobacter spp. were all over30%, and non-specific absorption of non-target bacteria was lower than3%. Interference assay showed that sensitivity of IMS is100times more sensitive than conventional direct plating method. Test using simulated samples of milk powder and feces showed that detection limit of IMS was101cfu/25g and101cfu/mL respectively, and the detection rate was improved.2) LAMP reaction system for detection of Cronobacter spp. was established. Each reaction mixture (total volume of25μL) contained2.5μL of6mM MgCl2,0.6mM dNTPs,0.8M Betaine,16pmol each of FIP and BIP primers,4pmol each of the F3 and B3primers, and2μL of DNA sample. And reaction for60min at58℃. After enrichment with IMS, LAMP showed higher specificity than PCR in detection of the ATCC29544strain of Cronobacter spp., and the detection limit of LAMP was101cfu/ml.3) Both LAMP and PCR successfully detected the3Cronobacter spp.-positive samples, which was consistent with results of national standard method.4) Of the209samples of infant formula milk powder in market,16were Cronobacter spp.-positive in IMS-LAMP test, while15were positive as shown by national standard method. The IMS-LAMP detection method showed the same result as that of national standard method, thus is similarly reliable, but it greatly reduced time required for detection.4. ConclusionThis study established the IMS technique to capture Cronobacter spp., and primers specific for the OmpA gene encoding the outer membrane protein of Cronobacter spp. were designed for LAMP analysis, and the parameters of reaction were optimized to ensure reliability and specificity of this method. LAMP assay were also performed for10other bacteria including E. coli, Salmonella, Shigella and some other pathogenic bacteria of the intestinal tract, as well as staphylococcus aureus, the results were all negative, indicating high specificity of this method. Detection limit of this method for bacterial culture was101cfu/mL, showing large advantage in detection sensitivity. The IMS-LAMP method we established for detection of Cronobacter spp. can be directly used for analysis of milk powder and other foods, and compared to conventional national standard method, it largely reduces time requirement, saving manpower and money. The IMS-LAMP method is simple, fast, performed under constant temperature, and requires only2-3h for detection of standard culture or26h for milk powder (national standard method requires6days). In addition, application of this method requires no expensive delicate instruments-a constant temperature water bath would be sufficient, and reaction and product testing can be performed in a single step, which is extremely simple and cost-efficient.In summary, the IMS-LAMP method we established for detection of Cronobacter spp. is highly sensitive, highly specific, simple and fast, and cost-efficient, shedding light on a new direction of development in Cronobacter spp. detection. This method has the potential to become a simple routine test, and is especially suited for grassroots inspection and quarantine institution. This method is of great significance in improving hygiene standard of food, ensuring food safety, and promoting international trade. |
PDF Full Text Request