Table 1.
Literature survey of the three critical aspects for proper bacterial quantification by qPCR. The papers analyzed were all published in 2021
| Scientific field | Purpose | Normalization strategy # | Calibration curve ### | Reaction efficiency ## | Reference |
|---|---|---|---|---|---|
|
| |||||
| Vaginal infections | To quantify G. vaginalis before and after exposure to Lactobacillus spp. | F | 2 | vi) | (He et al., 2021a, b) |
| To quantify Candida spp. and bacterial load in vaginal swabs | F | 2 | vi) | (McKloud et al., 2021) | |
| To quantify Gardnerella spp., Lactobacillus spp. and total bacterial population in vaginal swabs | E | 2 | i) | (Turner et al., 2021) | |
| To quantify the bacterial load and G. vaginalis in cervical fluid | D | 3 | vi) | (Kacerovsky et al., 2021) | |
| To quantify G. vaginalis and Lactobacillus spp. in mouse vaginal tissue | B | 4 | iv) | (Selis et al., 2021) | |
| To quantify Enterobacteriaceae, Staphylococcus spp., and Streptococcus spp. in vaginal swabs | F | 2 | vi) | (Oh et al., 2021) | |
| Wound infections | To quantify different bacteria in a polymicrobial biofilm | D | 2 | vi) | (Li and Wu, 2021) |
| To quantify Staphylococcus aureus in mice tissues | F | 4 | vi) | (Do Pham et al., 2021) | |
| Gastrointestinal infections/microbiota | To quantify Clostridioides difficile in a polymicrobial biofilm | B | 3 | vi) | (Normington et al., 2021) |
| To quantify bacterial load and nine bacteria in feces samples | B | 3 | vi) | (Tonon et al., 2021) | |
| To quantify Prevotella histicola in human duodenal biopsies | F | 2 | i) | (Balakrishnan et al., 2021) | |
| To quantify bacterial load in cecum content and feces | F | 3 | vi) | (Taibi et al., 2021) | |
| To quantify enterotoxigenic E. coli F4 in colon and ileal mucosal samples of piglets | F | 2 | vi) | (Rodríguez-Sorrento et al., 2021) | |
| To quantify Bacteroides vulgatus in fecal samples | F | 3 | ii) | (Maier et al., 2021a, b) | |
| To quantify fungal load in stool samples | B | 2 | vi) | (Boutin et al., 2021) | |
| To quantify bacteria and fungi in honeybee gut samples | F | 3 | vi) | (Callegari et al., 2021) | |
| Pulmonary infections | To quantify Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory samples from cystic fibrosis patients | F | 3 | vi) | (Pittet et al., 2021) |
| To quantify three bacteria associated with ventilator-associated pneumonia in dual-species biofilms with Candida albicans | F | 2 | i) | (Luo et al., 2021) | |
| To quantify bacteria in bronchoalveolar fluid samples | F | 3 | vi) | (Invernizzi et al., 2021) | |
| Oral biofilms | To determine the composition of polymicrobial biofilms | F | 3 | vi) | (Redanz et al., 2021) |
| To determine and quantify bacteria in dental caries | F | 2 | vi) | (Chen et al., 2021) | |
| To quantify bacteria in a polymicrobial biofilm | F | 3 | vi) | (Verspecht et al., 2021a) | |
| To detect and quantify bacteria in polymicrobial biofilms from saliva and dental plaque | B | 4 | vi) | (Oliveira et al., 2021) | |
| To quantify pathogenic bacteria in subgingival plaque and saliva samples | F | 2 | vi) | (Sereti et al., 2021) | |
| To quantify bacteria and fungi in a tooth model | F | 4 | i) | (Leelapornpisid et al., 2021) | |
| To quantify bacteria in a dual-species biofilm | B | 5 | vi) | (Millones-Gómez et al., 2021) | |
| To quantify bacteria in a polymicrobial biofilm | F | 3 | vi) | (Verspecht et al., 2021b) | |
| To quantify bacteria in a multi-species community and bacteria present in ligatures placed around teeth of mice | C | 2 | vi) | (Hoare et al., 2021) | |
| To quantify bacteria in a polymicrobial biofilm | F | 2 | vi) | (Chathoth et al., 2021) | |
| To quantify Porphyromonas gengivalis in oral samples | F | 2 | vi) | (Franciotti et al., 2021) | |
| To quantify two bacteria in oral samples and colon tissue | F | 2 | vi) | (Pignatelli et al., 2021) | |
| To quantify microbial load in saliva samples | F | 2 | vi) | (Marotz et al., 2021) | |
| To quantify four bacteria in subgingival samples | F | 2 | v) | (Cuenca et al., 2021) | |
| To quantify bacterial load, and two different bacteria in periapical tissue | F | 2 | vi) | (Bordagaray et al., 2021) | |
| To quantify Streptococcus mutans and Candida albicans in oral samples | F | 2 | vi) | (Yang et al., 2022) | |
| Soil microbiota | To quantify bacterial and fungal load and Fusarium oxisporum in soil samples | F | 6 | v) | (Zhu et al., 2021a, b) |
| To quantify bacterial and fungal load | F | 2 | i) | (Ammitzboll et al., 2021) | |
| To quantify ammonia-oxidizing archaea and bacteria in soil samples | F | 3 | v) | (Dai et al., 2021) | |
| To quantify Streptomyces bottropensis and Brevibacillus laterosporus in soil samples | F | 3 | v) | (Li et al., 2021a, b, c) | |
| To quantify bacterial load in wheat root samples | F | 3 | v) | (Usyskin-Tonne et al., 2021) | |
| To quantify ammonia-oxidizing archaea and bacteria in soil samples | F | 3 | v) | (Samaddar et al., 2021) | |
| To quantify bacterial and fungal load in soybean soil samples | F | 3 | v) | (Gao et al., 2021a, b) | |
| To quantify Calonectria ilicicola in soybean soil samples | F | 2 | vi) | (Ochi and Kuroda, 2021) | |
| To quantify ammonia-oxidizing archaea and bacteria in soil samples | F | 3 | vi) | (He et al., 2021a, b) | |
| To quantify F. oxysporum, bacterial and fungal load, ammonia-oxidizing archaea and bacteria in soil samples | F | 3 | ii) | (Liu and Zhang, 2021) | |
| To quantify bacterial load, ammonia-oxidizing archaea and bacteria in soil samples | F | 3 | vi) | (Li et al., 2021a, b, c) | |
| To quantify bacterial load in soil samples | F | 3 | vi) | (Nogrado et al., 2021) | |
| To quantify bacterial load in soil samples | F | 2 | v) | (Han et al., 2021) | |
| To quantify bacterial load in dry soil samples | F | 3 | ii) | (Zhao et al., 2021) | |
| To quantify bacterial load in soil samples | F | 3 | ii) | (Wang et al., 2021a, b) | |
| To quantify bacterial load in soil samples | F | 3 | ii) | (Zhu et al., 2021a, b) | |
| To quantify bacterial load in soil samples | F | 3 | vi) | (Zhang et al., 2021a, b) | |
| To quantify Brevibacillus laterosporus and S. bottropensis in soil samples | F | 3 | v) | (Li et al., 2021a, b, c) | |
| To quantify ammonia-oxidizing archaea and bacteria in soil samples | F | 3 | vi) | (Wei et al., 2021) | |
| To quantify ammonia-oxidizing archaea, ammonia-oxidizing bacteria, Nitrobacter and Nitrospira spp. in soil samples | F | 3 | vi) | (Yin et al., 2021) | |
| To quantify Rhizoctonia solani and Rhizoctonia solani AG1-IB in lettuce and soil samples | D | 2 | i) | (Wallon et al., 2021) | |
| To quantify bacterial load in soil samples | B | 2 | vi) | (Alvarez et al., 2021) | |
| To quantify F. oxysporum f. sp. cubense, Fusobacterium solani and Aspergillus fumigatus in soil samples | F | 3 | vi) | (Yuan et al., 2021) | |
| To quantify bacterial and fungal load, Ralstonia solanacearum and F. oxysporum f. sp. Lycopersici in soil samples | F | 6 | vi) | (Deng et al., 2021) | |
| To quantify Azospirillum brazilense in soil samples | F | 1 | iii) | (Urrea-Valencia et al., 2021) | |
| To quantify Pseudomonas protegens and Bacillus subtilis in soil samples | F | 3 | ii) | (Zhang et al., 2021a, b) | |
| Bacterial contamination on surfaces | To quantify Lactobacillus delbrueckii subsp. Bulgaricus in surface samples | F | 2 | i) | (Wang et al., 2021a, b) |
| To quantify four Staphylococcus spp. in hospital surfaces | F | 2 | vi) | (Gismondi et al., 2021) | |
| To quantify Candida auris in skin swabs and hospital surfaces | F | 4 | vi) | (Sexton et al., 2021) | |
| Food quality control / Foodborne pathogens | To quantify three Lactobacillus spp. in sourdough bread | F | 2 | i) | (Baek et al., 2021) |
| To quantify Lactobacillus spp. in fermented dairy products samples | F | 2 | i) | (Yang et al., 2021) | |
| To quantify Brucella spp. in milk and cheese samples | F | 2 | vi) | (Marouf et al., 2021) | |
| To quantify seven proteolytic Pseudomonas spp. in raw milk samples | F | 1 | iii) | (Maier et al., 2021a, b) | |
| To quantify Nucleospora cyclopteri in fish and blood samples | F | 2 | i) | (Naung et al., 2021) | |
| To quantify different bacteria in meat samples | F | 2 | i) | (Bahlinger et al., 2021) | |
| To quantify Colletotrichum acutatum in olive fruit samples | F | 3 | v) | (Azevedo-Nogueira et al., 2021) | |
| To quantify Lysteria monocytogenes in meat samples | F | 1 | iii) | (Labrador et al., 2021) | |
| To quantify Shiga toxin-producing Escherichia coli in meat samples | F | 2 | vi) | (Rey et al., 2021) | |
| To quantify Campylobacter coli in meat samples | F | 2 | i) | (Lazou et al., 2021) | |
| To quantify Brochothrix thermosphacta in fish samples | F | 2 | i) | (Bouju-Albert et al., 2021) | |
| To quantify Streptococcus iniae in fish samples | F | 2 | i) | (Torres-Corral and Santos, 2021) | |
| To quantify Cronobacter sakazakii in spiked powdered infant formula samples | F | 2 | i) | (Gao et al., 2021a, b) | |
| To quantify four Campylobacter spp. in meat samples | F | 2 | i) | (Vizzini et al., 2021) | |
| To quantify Salmonella spp. in poultry floor dust | F | 1 | i) | (Ahaduzzaman et al., 2021) | |
| To quantify six bacteria genera and four bacteria phyla in milk samples | F | 2 | vi) | (Sanjulián et al., 2021) | |
| Waterborne pathogens | To quantify bacteria in polymicrobial biofilms present in a drinking water distribution system | C | 3 | i) | (Fu et al., 2021) |
| To quantify several enteric opportunistic pathogens in influent and recycled water | F | 2 | i) | (Drigo et al., 2021) | |
| To quantify Enterococcus spp. and Salmonella spp. in water, sand, and sediments samples | F | 3 | ii) | (Li et al., 2021a, b, c) | |
| To quantify Helicobacter pylori and Legionella spp. in filtered water samples | F | 5 | vi) | (Ribes et al., 2021) | |
| To quantify pathogenic fungi, enteric and opportunistic pathogens | F | 3 | ii) | (Hu et al., 2021) | |
| To quantify several bacteria in water samples | F | 1 | iii) | (Ambili and Sebastian, 2021) | |
| Other fields | To detect and quantify bacteria in breast implant samples | E | 2 | vi) | (Crowe et al., 2021) |
| To quantify bacteria in bovine digital dermatitis | B | 3 | v) | (Caddey et al., 2021) | |
| To quantify bacterial load in livestock fecal manure samples | F | 3 | vi) | (Wongsaroj et al., 2021) | |
| To quantify Zobellia genus in macroalgae surface | F | 2 | i) | (Brunet et al., 2021) | |
| To quantify Campylobacter fetus subsp. fetus and Salmonella enterica subsp. enterica serovar Typhimurium in bovine endometrial cells | F | 4 | vi) | (Muzquiz et al., 2021) | |
| To quantify five bacteria in cosmetic cream samples | F | 2 | i) | (Bermond et al., 2021) | |
| To quantify bacterial and fungal load in dust samples | F | 4 | vi) | (Haines et al., 2021) | |
| To quantify bacterial load in skin swabs, and skin samples from patients with atopic dermatitis | F | 2 | vi) | (Edslev et al., 2021) | |
| To quantify bacterial load in milk, feces and blood samples from healthy cows and cows suffering from bovine mastitis | A & B | 6 | vi) | (Scarsella et al., 2021) | |
#
Normalization strategy—A—An exogenous control was used; B- Initial gDNA concentration normalization among samples; C—gene copy numbers were normalized by the filtrate membrane surface area / ligature length; D-16 s rRNA gene (internal control) was used; E—an exogenous control was used only in the qPCR run. F- No normalization strategy was used
##
Calibration curve—1—Calibration curve performed using gDNA isolated from pure cultures with different initial concentrations, or pure cultures mixed with sample background of interest; 2—calibration curve performed with gDNA dilutions from one sample; 3—calibration curve performed by cloning a gene into a plasmid to determine absolute copy numbers by performing dilutions of the sample; 4-it is not clear whether the calibration curve was performed using dilutions of one extraction or different extractions from different bacterial concentrations; 5—standard curve was mentioned but it was not explained how it was constructed. 6 -No standard curve was mentioned
###
Reaction efficiency—i—gDNA from one sample; ii—dilution of one sample with a plasmid containing the gene of interest; iii—gDNA extracted from samples with different concentrations of bacteria; iv—it is not clear whether qPCR primer efficiency was determined by using dilutions of one gDNA sample or from gDNA samples isolated from samples with different initial bacteria concentration; v—considered but the procedure to determine the efficiency was not mentioned; vi—not mentioned