Standard bacterial strains
The standard strains for this experiment focus on the main intestinal pathogens. Standard strains of 7 bacterial species selected to develop the assay were purchased from Shanghai Bioplus Biotech Co., Ltd (Shanghai, China), and their sources are shown in Table 1. The standard strains were identified by VITEK 2 Compact automatic bacterial identification and analysis system from the microbiology laboratory of the Quarantine Service (Gansu Provincial Center for Disease Control and Prevention (GSCDC), Lanzhou, Gansu province, China).
Collection and treatment of housefly samples
A total of 1060 houseflies were randomly collected from four different environments in Lanzhou of China, including residential area (n=380), slaughterhouse (n=330), garbage transfer station (n=200), near hospital (n=150) from 2016 to 2017. Ten houseflies per group were packed into autoclaved triangular flasks, 10 ml physiological saline was added, and washed by shaking for 10 min for subsequent DNA extraction.
DNA was extracted from the overnight cultures of the bacteria using DNA extraction kit for Gram-negative bacteria (ABT) according to the manufacturer’s instructions. The extracted DNA was stored at -20 ºC until the subsequent analysis. Briefly, 1 ml of the overnight bacterial culture was centrifuged for 5 min at 10000 rpm, the supernatant was discarded. 1 ml physiological saline was added in the precipitate, and the above mixture was shocked to disperse bacteria and then centrifuged for 5 min at 10000 rpm, the supernatant was discarded. 200 μl of sterilized ddH2O was then added, mixed thoroughly and the supernatant was discarded after centrifuging for 3 min at 13000 rpm. After adding 50 μl nucleic acid extract into the bacteria precipitate, mixed thoroughly and centrifuged instantaneously, the hanging wall liquid was flung to the bottom of the EP tube. The EP tube containing the bacteria solution was heated in water bath at 100 ºC for 10 min, and then centrifuged for 10 min at 13000 rpm, the supernatant was used as the DNA template in subsequent amplification experiments.
Primer and probe design
The 16S RNA was found out to be highly conservative, according to the literature  and the GenBank database. The sequence alignment of the ribosome 16S RNA of 7 bacterial species (S. aureus, S. flexneri, A. caviae, V. vulnificus, S. enterica, P. vulgaris, Y. enterocolitica) was carried out. Universal primer (RLB-F, RLB-R) for PCR amplification of genomic DNA samples used in PCR-RLB hybridization assay, species-specific probe and universal probe (Catch-all) were designed using DNAStar and Primer premier software. To test for theoretical specificity, all the primers and probes used were aligned with the sequence databases of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLASTn). Universal primers were labelled at the 5’-end with biotin to allow PCR products to be detected by hybridisation with a streptavidin–peroxidase substrate in the RLB assay. All probes were labelled at the 5’-end with an amine group to facilitate covalent linkage to nylon membranes and to allow membranes to be stripped and reused repeatedly. The primers and probes were synthetized by Sangon Biotech Company, China (Table 2).
Genomic DNA (of the standard strains or samples) was added to a reaction mixture (final volume of 25 μl) containing 40M of both primer RLB-F and RLB-R. PCR amplification was performed in an automatic DNA thermocycler (Eppendorf). The reaction was incubated at 94 ºC for 5 min to denature genomic DNA and the thermal cycle reaction programme was: 30 s at 94 ºC, 30 s at 63 ºC and 45 s at 72 ºC for 35 cycles with a final extension step of 72 ºC for 10 min. Samples were held at 12 ºC until analysis.
The RLB protocol was performed as described previously . Briefly, a Biodyne C blotting membrane (BNBCH5R, Pall BioSupport) was activated at room temperature by incubating in 16% EDAC (E7750, Sigma) for 10 min, then washed in distilled water, and placed in a MN45 miniblotter (FZB, Germany). Species-specific oligonucleotide probes were diluted to different concentrations (25, 50, 100, 200, 500, 800, 1000 μM) in 500 mM NaHCO3 (pH 8.4), added to the miniblotter slots, and incubated for 2 min. Then, the membrane was incubated in 100 mM NaOH for 10 min and rinsed with demineralized water at 60 °C for 5 min in 2 × SSPE/0.1 % SDS. The membrane was then placed perpendicular to the probe orientation in the miniblotter. Twenty microliters of each PCR product was diluted in 2×SSPE with SDS 10% w/v to a final volume of 150 μl, heated to 99 °C for 10 min, and then cooled immediately on ice. The denatured PCR products were then added to the slots in the miniblotter and incubated for 60 min at 60 °C, and the membrane was washed twice at 60 °C for 10 min in 2×SSPE with SDS 0.5 %. Additionally, the membrane was treated at 42 °C for 60 min with peroxidase-labeled streptavidin diluted 1:4000 in 2×SSPE/0.5 % SDS and washed twice at 42 °C for 10 min in 2×SSPE/0.5 % SDS and twice at room temperature for 5 min in 2×SSPE. Finally, chemiluminescence detection was performed according to standard procedures (Amersham).
Specificity and sensitivity of RLB
For specificity studies, DNA was extracted from standard strains (Table 1) using a DNA extraction kit for Gram-negative bacteria (ABT) according to the manufacturer’s instructions, and was tested against all probe sets.
To assess RLB sensitivity, the genomic DNA content of the standard strains was determined by nucleic acid concentration meter (NanoDrop ND-2000). Serial ten-fold dilutions of genomic DNA (starting at 100 ng/µl) were prepared into 10-1-10-12 in distilled water and then used as template for the RLB sensitivity analysis.