Development of a simple, rapid multiplex PCR tool kit by using 16S rRNA gene for the identi ﬁ cation of faecal and non-faecal coli forms in the drinking waters

A multiplex method for the detection of faecal and non-faecal coliforms in drinking water was developed using three primers from the V2, V3 and V9 variable regions of 16S rRNA gene. 194F, 474F and 1436R are the three primers designed for speci ﬁ c ampli ﬁ cation of V2, V3, V9 hyper variable regions of 16S rRNA gene. Multiplex PCR allowed for differentiation of the total coliform from faecal coliform by speci ﬁ c amplicons: 1,285 bp of amplicon is speci ﬁ c for 6 non-faecal coliform genera and 1,009 bp of amplicon is speci ﬁ c for faecal coliform ie. E. coli . If the drinking water was contaminated with both faecal and non-faecal coliforms then two amplicons of 1,285 bp and 1,009 bp by combination of three primers are observed. The multiplex PCR assay based on 16S rRNA gene should be a bene ﬁ cial tool kit for the rapid identi ﬁ cation of the total coliforms in the large number of water samples compared with traditional methods. Results can be acquired within 3 hrs of time as compared with classic method of MPN (3 – 4 days). This assay will be useful in diversi ﬁ cation and detection of seven genera of total coliforms by using variable regions of 16S rRNA.


GRAPHICAL ABSTRACT INTRODUCTION
Microbial safety of drinking water was assessed by the establishment of the presence of the microbial indicators like non-coli and faecal coliform bacteria such as Escherichia coli (E. coli). Faecal contamination in water was monitored by the existence of E. coli and entails that presence of pathogenic viruses, protozoa, bacteria, (Environment Agency ). Faecal contamination of water bodies may produce diarrheal illness and cause human mortality (Levy K. 2015). In the developing world, the major source of human illness is due to water related diseases (Magana-Arachchi, D. N et al. 2020). These vulnerable diseases to humans are caused due to drinking of untreated and contaminated water. E. coli O157:H7 is the most common type of Shiga toxin-producing Escherichia coli (STEC), but other types exist, which includes non O157; H7 strains include: O26:H11, O103:H2, O145:H28, and O111:H8, produces shiga toxin in the environmental waters, which are the major reservoiurs of infectious E. coli (Bonetta et al. , Iweriebor, B. C et al. 2015).
Global health issues have arisen due to waterborne diseases. Hence rapid identification of indicators of faecal contaminants, possibly the human pathogens is very critical in environmental samples. The identification and enumeration of bacteria from water are relevant, as it contributes to healthy public consumption of safe drinking water (Kaestli, M et al. 2019). Therefore, numerous methods for rapid detection and quantification of water quality indicators and waterborne pathogens have been established.
Traditional approaches based on cultures are laborious, time-consuming. A rapid, simple protocol for the direct detection of microbes is therefore needed to be established (Cruz, C. D. et al. 2012).
Compared to the conventional approach, multiplex PCR is a valuable, quick and secure substitute for microbiological identification of drinking water organisms. For the concurrent amplification of target sequences in a single PCR, multiplex PCR (MPCR) uses one or more template and several sets of primers (Molina et al. 2015). For the differentiation and recognition of bacterial species, multiplex PCR uses several templates and special primers (Bourque et al. 1993;Geha 1994;Yoon et al. 1996b

Bacterial strains
Isolates used in the present study were procured from Microbial Type Culture Collection (MTCC), Chandigarh, India are tabulated in Table 1.

Study location
Mahabubnagar is one of the drought districts in Telangana state. The geographical area of the district is 18,432 Sq.  DNA ladder (Figure 1) was used to classify the product bands. Using a PCR purification Kit (Bangalore Genei, India), the amplified product was purified and sequenced at Bioserve Biotechnologies Pvt, Hyderabad. Further, the 16S rRNA gene sequence was deposited in the NCBI Gen-Bank and accessions numbers were allocated in Table 3.

Multiplex PCR
The multiplex PCR reaction mixture consisting of 25 μl of KAPA SYBR ® FAST qPCR Master Mix (2X) Kit (Sigma Aldrich, City, India) or subsidieary in India. The PCR conditions are similar for 1 μl 5 pmol of forward primers 194F, 474F and 1 μl 5 pmol of reverse primer 1436R, 1 μl 30-50 ng DNA template and finally 12 μl PCR grade water for a total volume of 50 μl were used for the amplification       In order to validate the designed primers standard cultures of coliforms (7 genera) were procured from MTCC.
Later the 1,285 bp gene sequence was confirmed and deposited in NCBI with accession No. (MW029934-MW029940).
Meanwhile the non-coliforms by 16S rRNA and biochemical, phenotypic tests (Pindi PK et al. 2013), were found to be correctly identified with the newly designed primers.
Thus accuracy of the primers for coliforms in the present study was found to be 99%.

DISCUSSION
Water-borne disease outbreaks continue to be a significant concern for global public health providers, claiming millions

DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.