Simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms by the reverse line blot hybridization assay

Background: As a widespread health pest, flies can carry more than 100 kinds of pathogenic microbes to threat human health, resulting in a wide range of disease infection and transmission. The aim of this study was to develop a sensitive, reliable and rapid method for the simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms, in order to effectively prevent and control fly-borne bacterial diseases. Results: PCR-RLB method could directly and accurately detect fly-borne bacteria species corresponding of 7 species-specific probes. At the same time, the membrane binding oligonucleotide species-specific probes prepared in RLB detection technology can be reused for detection of bacteria after washing with 0.5 M EDTA, which greatly improves the detection efficiency. In 106 groups of samples, the numbers of samples carrying seven different bacterial strains were 2 ( S. aureus ), 52 ( S. flexneri ), 0 ( A. caviae ), 3% ( V. vulnificus ), 56 ( S. enterica ), 1 ( P. vulgaris ) and 33 ( Y. enterocolitica ), respectively. Their proportions of 7 bacterial strains carried by houseflies were 1.23% ( S. aureus ), 32.1% ( S. flexneri ), 0% ( A. caviae ), 1.85% ( V. vulnificus ), 34.57% ( S. enterica ), 0.62% ( P. vulgaris ) and 20.37% ( Y. enterocolitica ), respectively. It was found that the worse the hygienic condition, the higher the bacteria carrying rate of houseflies was. S. enterica , S. flexneri and Y. enterocolitica accounted for the overwhelming majority of the seven pathogenic strains carried by houseflies from four different environments in Lanzhou. This indicated that houseflies played an important role in the transmission of intestinal diseases, which was mainly related to the breeding and reproduction of houseflies in feces, carrion and food. S. aureus was carried by houseflies in the hospital area indicates that hospitals should do well in killing and controlling flies and further strengthen the prevention and control of fly-borne bacterial diseases. Conclusion: The RLB assay appeared to have potential clinical

simultaneous detection of fly-borne bacterial species.
There are a wide variety of flies, among which only a few species are the most common in the human family and around the farm, including housefly, stomoxys calcitrans, lucilia sericata, sarcophagidae and so on. Flies feed and reproduce in animal feces, organic wastes and carcasses, being one of the important threats to human health [9][10][11][12][13][14][15][16][17][18]. On February 24, 2005, the Science Times reported that flies spread of Enterohemorrhagic E.
coli and avian influenza in Japanese. In China, flies are also included in the key prevention and control target of insect-borne diseases.
Insect-borne bacterial disease detection is mainly based on traditional bacterial culture and isolation. Identification of each strain takes at least a week or so. This method is time and labor-consuming and has strict requirements on the laboratory environment, which leads to the isolation and cultivation of bacteria cannot be carried out in areas without large laboratories, and seriously affect the prevention and treatment of insect-borne diseases. The rapid and efficient detection of insect-borne bacterial diseases has become a hot field in the prevention and control of insect-borne diseases. Reverse line blot (RLB) is a sensitive and high-throughput detection method, which can simultaneously detect various pathogenic microorganisms carried by insects. Its essence is the combination of PCR product single chain and species-specific probe to determine the difference of the amplified sequence. PCR-RLB technology has high sensitivity and specificity. It can distinguish various strains of mixed infection, and even identify species. So it was widely used in the detection of various diseases, such as Kaufhold et al. (1994) for the first time to use PCR-RLB in serotype identification of streptococcus [ 19]; O'Sullivan et al. (2011) used PCR-RLB technology to analyze the drug resistant strains of Staphylococcus aureus [20]; Nijhof et al. (2005) applied this method to analyze four species of Taylor in Africa [21].  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 ddH 2 O 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 [22] 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. 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 streptavidinperoxidase 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). Table 2 Sequence and concentration of Primers and probes used in the study

RLB Hybridization
The RLB protocol was performed as described previously [23]. 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

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. Y. enterocolitica-2 probe (Fig. 2).

Selection of probes and primers
Initial evaluation experiments revealed that the optimal primer concentration of RLB-F and RLB-R was 50 µM; the optimal probe concentration was 50 µM for Catch-all, S.aureus-2 and S.flexneri-2 probe, and 100 µM for other selected probes (Table 2).

Specificity Of RLB
All selected probes bound only to their respective target sequence, resulting in the recognition of individual bacterial species. The nucleotide probes did not show any crossreaction with water used as a blank control. The catch-all probe specifically detected any standard strains present. Each standard strain was identified by two oligonucleotide probes: the catch-all probe and species-specific probes for either 7 bacterial species (Fig. 3).

Sensitivity Of RLB
The  Fig. 6 can not only clearly display the bacterial carrying status of samples, but also analyze the carrying and carrier rate of bacteria corresponding to different probes in different environments. The detail of the analysis results was shown in Fig. 7 and Table 3. Table 3 The

Discussion
In all kinds of bacterial detection experiments, the first thing is to clarify the source of bacterial strain, and confirm that bacterial strain did not mutate, so all the standard strains used in this study have been identified. Sequence alignment of 16S RNA gene sequences of 7 standard bacterial strains with clear background was carried out, universal primers and species-specific oligonucleotide probes were successfully designed. The target sequences of all bacterial strains were successfully amplified by using universal primers for PCR amplification. And species-specific probes (S.aureus-2, S.flexneri-2, A.caviae-2, V. vulnificus, S. enteric, P. vulgaris-1, Y. enterocolitica-2) aimed at the target gene sequences were successfully screened out. The common primers were used for PCR amplification. The target sequences of all strains were successfully amplified, and the target sequences were successfully screened out. A simultaneous detection method of 7 bacteria species by PCR-RLB was successfully established. The sensitivity of the PCR amplification products with different concentration prepared by serial ten-fold dilutions was tested. The results showed that the sensitivity of PCR-RLB was significantly higher (about 100 times) than that of PCR, which is consistent with literature reports [23][24][25][26][27][28][29].
In summary, a high specificity and sensitivity method for the simultaneous detection of 7 bacteria strains by PCR-RLB was successfully established in this study. This method was The sensitivity of the PCR amplification products with different concentration prepared by serial ten-fold dilutions was tested, and the results showed that the sensitivity of PCR-RLB was significantly higher (about 100 times) than that of PCR. This method was then used to enterocolitica.

Figure 2
The selection of probe. Oligonucleotides probes are applied in horizontal rows and

Figure 7
The statistical result of detection bacteria carried by houseflies collected from four different environments of Lanzhou using PCR-RLB method.